The GM130 and GRASP65 Golgi Proteins

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The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment
Pierfrancesco Marra*, Tania Maffucci*, Tiziana Daniele*, Giuseppe Di Tullio*, Yukio Ikehara, Edward K. L. Chan, Alberto Luini*, Gala Beznoussenko*, Alexander Mironov* & Maria Antonietta De Matteis*
*Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA e-mail: demattei@cmns.mnegri.it
Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incoming IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face.
he Golgi apparatus has a complex architecture composed of stacks of flat cisternae connected in a ribbon-like fashion by tubular reticular areas. How such an organization is achieved at the cis-Golgi face and maintained through the Golgi in spite of the continuous flux of membranes remains a central problem in the biology of transport. Two classes of protein complexes are thought to be involved in morphogenesis of the Golgi complex, possibly through a coordinated action: the Golgi spectrinactin skeleton and the Golgi matrix proteins. The spectrin skeleton assembles on Golgi membranes in a fashion dependent on the activity of the small GTPase ARF (ADP-ribosylation factor) and on the level of phosphatidylinositol 4,5-bisphosphate1,2. The spectrin skeleton facilitates incorporation of endoplasmic reticulum (ER)derived membranes into the cis-Golgi1; it may also control the structure of Golgi stacks, on the basis of the probable similarity of the known role of spectrin at the plasma membrane3. The Golgi matrix proteins4,5 were originally identified as a set of insoluble Golgi proteins, and include GM130 and GRASP65. These proteins are involved in postmitotic reassembly and stacking of the Golgi cisternae68 and can assemble in a structure reminiscent of the Golgi even in the absence of Golgi resident proteins suggesting that they can act as primary scaffold components underpinning the Golgi architecture9. Remarkably, GM130, GRASP65 and other Golgi matrix proteins are located primarily at the cis-pole of the Golgi stacks4,6, where they probably function in the incorporation of the ER-derived membranes into the Golgi. The membranes that reach the cis-Golgi are tubular clusters that constitute the ERGolgi intermediate compartment (IC)10. The biogenesis, maintainance, and identity of the IC have been and are objects of intense study. Current theories suggest that the IC may be considered either as an outgrowth of the ER11,12, as a part of the cisGolgi network (CGN)13, or as a separate compartment1417. In the latter case, the IC might be viewed as a stable compartment15,16 or as a transient compartment made up of transport intermediates operating between the ER and the Golgi14,17. The difficulties in reconciling
these apparently contrasting views and in defining the boundary of the IC result largely from the disperse distribution and pleomorphic organization of this compartment, and from its dynamic behaviour10. Even the molecular composition of the IC elements is highly heterogenous18 and includes proteins continuously cycling between the ER and the Golgi complex. However, by combining the broadest definition of the ERGolgi IC (consisting of the membranes interposed between the ER and the Golgi stacks) with the information derived from the dynamics of proteins traversing this compartment (which includes cargo proteins19,20, recycling proteins21, and the coat complexes COPI and COPII2224), it is possible to distinguish three layers in the IC. The first layer includes the ER exit sites (ERES, or transitional ER), marked by COPII, and made up of rather stationary elements22,24. The second layer consists of tubular clusters that move long distances, travelling in centripetal direction along microtubules; this layer contains COPI, but not COPII, and forms what we define from here onwards as IC. The third layer includes the CGN, which consists of tubular clusters juxtaposed to the cis-Golgi cisterna (which in turn contain the cisGolgi markers GM130, GRASP65, mannosidase I, and Helix Pomatia (HP) lectin-binding proteins25). According to the view of the IC as a transient compartment, these three layers correspond to three stages of the IC membrane lifespan: origin at the ERES via a sorting process mediated by the SAR1pCOPII complex; motordriven translocation towards the Golgi along microtubules and concomitant loss of ER-recycled components; and final incorporation into the Golgi complex. Although the first two stages have been extensively studied and their molecular machineries satisfactorily elucidated, the process by which the IC membranes are incorporated into the cis-Golgi remains elusive. To gain insight into the details of this final step, we investigated how Golgi-structural components such as the matrix proteins GM130 and GRASP65 associate with the incoming IC membranes. We find that IC elements acquire these Golgi components before contacting the cis-pole of the central Golgi area, at the level of a
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NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com
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Figure 1 GM130 cycles between the Golgi complex and peripheral compartments via tubules. A, Distribution of GFPGM130 in COS7 cells. Note that GFPGM130 localizes not only to the central Golgi area, but also to peripheral puncta and tubules. BF, Dynamics of GFPGM130 in living COS7 cells. Time is given in min.sec. B, Snapshots from Supplementary Information movie 1. An anterograde tubule originating from a puncta (01.18) elongates towards the Golgi area (01.20), transiently connects the donor and acceptor compartments (01.21), detaches from the puncta (01.22), and finally disappears into the central Golgi area (01.24) while the peripheral puncta remains apparently unchanged compared with the first frame (compare 01.18 with 01.24). C, Snapshots from Supplementary Information movie 2. Frames from 00.09 to 00.30 show a retrograde tubule originating from the Golgi area (asterisk), detaching from the latter, moving towards, and fusing with a peripheral puncta (indicated with a filled arrowhead). The trajectory of the movement of the puncta is indicated with a dashed line. The same puncta, receives a tubule from a neighbouring puncta (00.34). The tubule transiently connects the two puncta before fusing and disappearing into the acceptor puncta (00.37). D, GM130 retrograde tubules mediate the fluorescence recovery after pho-
tobleaching (FRAP) of the peripheral puncta. The peripheral area included between the two red profiles and including puncta and tubules was bleached (one bleaching round of 100 iterations, frame 00.00) and the FRAP of this area was recorded over 2 min after the bleaching. Note that numerous tubules emanate from the Golgi area (1.08, 1.31), generate puncta that are interconnected by tubules (1.56) and finally appear as separate puncta (1.58). E, Bleaching of the Golgi area induces fluorescence loss of the GM130-positive puncta. Five bleaching rounds (total time, 3 min) of the Golgi area (indicated by the red profile in a) induced almost complete loss of fluorescence in the puncta (b). F, Bleaching of the puncta and tubules induces fluorescence loss of the Golgi. The peripheral area, delimited by the red profile (a and b), and then the area including puncta and tubules and delimited by the blue profile (a and d), were repeatedly bleached (five bleaching rounds over 16 and 10 min, respectively), and the fluorescence of the Golgi area was measured in c and e, respectively. It is evident that the bleaching of the blue area (d, e), but not that of the red area (b, c) reduced the fluorescence in the Golgi area (compare e with c). Scale bars: A, 4 m; B, 2 m; C, 4 m; D, 8 m; E, 6 m; F, 6 m.
specialized subdomain of the IC that we define as late IC (L-IC); this is where the transition between IC and cis-Golgi takes place.
Results
GM130 cycles between the Golgi complex and peripheral transport stations via membranous tubules. We examined the dynamics of GM130 in living cells by analysing a GFPGM130 chimaera in COS7 fibroblast and NRK epithelium cells. This chimaeric protein behaves exactly like the endogenous GM130 under all the conditions tested (see Methods), and becomes incorporated into the Golgi matrix to the same extent as GM130 (data not shown). When imaged in living COS7 cells, GFPGM130 localized not only at the central Golgi area, as expected4, but also on two types of peripheral structures, which we define as puncta and tubules (Fig. 1A). The GFPGM130-containing puncta were distributed throughout the
cytoplasm but were more concentrated around the Golgi area, had an average apparent size of around 0.8 m (ranging from 0.3 to 3 m), and were highly mobile. They moved with speeds ranging from 0.2 to 0.8 m s1 and a directionality that suggests microtubule-based motility, that is, towards the central Golgi or other puncta, which they often eventually joined. While moving, they received and emitted tubules. The tubules were more transient and mobile than the puncta. They elongated and moved in centrifugal, centripetal and lateral directions. The shape, distribution and movement patterns of puncta and tubules were highly complex. Sometimes the tubules originated from GFPGM130-labelled peripheral puncta, projected centripetally towards the Golgi area while still connected to the original puncta, and after reaching this area, disappeared into the Golgi mass (Fig. 1B and C; and Supplementary Information movies 1 and 2). Other tubules extended centrifugally from the central area towards the peripheral
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Figure 2 GM130-positive peripheral structures have compositional features of an IC subdomain. A, GM130 does not colocalize with cis- or medial-Golgi markers on puncta and tubules. NRK cells (a, b, e, f), RBL cells (c, d) and COS7 cells (g, h) were labelled with anti-GM130 antibodies (a, c, e, g) and with anti-mannosidase I (b), anti-mannosidase II (f), anti-giantin (h) antibodies, or with FITC-conjugated HP lectin (d). Note that the GM130-positive puncta and tubules (filled arrowheads) are not labelled by anti-mannosidase I or II, anti-giantin antibodies, or HP lectin (empty arrowheads). B, C, GM130-positive tubules and puncta identify a subdomain of the IC. B, GM130 colocalizes with GRASP65 and IC markers, but not with ERES markers on puncta and tubules. COS7 cells were double stained with anti-GM130 (a, c, e, g, i) and with anti-GRASP65 (b), anti KDEL-R (d), p115 (f), SEC13 (h) or COP (j) antibodies. Note that the GM130-positive puncta and tubules (filled arrowheads) contain GRASP65 and markers of the IC (KDEL, p115,
COP), but almost exclude SEC13 (a marker of the ERES, empty arrowheads). Note also the IC markers and SEC13 are present on many peripheral puncta (some of which are indicated by filled arrows) that are devoid of GM130 (empty arrows). C, COS7 cells were transfected with GFPGM130 (green) and then processed for immunofluorescence, and labelled for an ERES marker (SEC31, blue) and for an IC marker (ERGIC53, red). The boxed area is enlarged in the insets. Four populations of elements can be distinguished: one labelled exclusively by SEC31 (blue), one by SEC31 and ERGIC53 (purple), one labelled exclusively by ERGIC53 (red), and one by ERGIC53 and GM130 (yellow). The last population corresponds to the IC subdomain identified by GM130 (L-IC), whereas the red elements represent the E-IC (see text). Note that in many cases, the overlap of the stainings of SEC31 and ERGIC53 is not complete and the blue (SEC31) and red (ERGIC53) elements are not concentric. Scale bars, 4 m.
puncta. The retrograde direction of these tubules was verified studying the fluorescence recovery after photobleaching (FRAP) of the peripheral puncta: the fluorescence recovery of the bleached puncta was clearly mediated by long tubules originating from the central Golgi and extending towards the periphery (Fig. 1D). Other tubules were seen to emanate from peripheral puncta, elongate towards neighbouring or distanct puncta, thus connecting peripheral puncta with each other, and finally disappear into the acceptor structures. In some cases the tubules did not establish continuity between two structures, but apparently detached from the puncta from which they originated and then moved towards their destination (Fig. 1C and Supplementary Information movie 2). The average tubule life span was 5 s (ranging from 2 to 11 s), and length was 6 m (ranging from 3 to 12 m). The tubules were collinear with microtubules, and were dependent on the presence of an intact microtubule system, as they disappeared in cells treated with nocodazole (data not shown). Thus, both the tubules and the puncta are mobile, but tubules are more transient and dynamic; in fact, puncta behave
as slowly moving platforms that emit tubules. Overall, the complex dynamics of these elements suggest that tubules create a permanent semistable system of interconnections between peripheral puncta and between puncta and the GM130 central membrane pool. The result is a spatially dispersed, but dynamically well connected, membranous system. To investigate the dynamic relationships between the peripheral and central pools of GM130, the Golgi area or the puncta and tubules were bleached repeatedly, after which the GFPGM130positive structures were examined using the FLIP (fluorescence loss in photobleaching) technique. Five bleaching rounds (total time 3 min) of the Golgi area induced almost complete loss of fluorescence in the puncta (Fig. 1E), and five bleaching rounds (total time 10 min) of the puncta and tubules significantly decreased the fluorescence in the Golgi area by 70% (Fig. 1F, d and e). These data indicate that the pools of GM130 located at the Golgi and at peripheral puncta communicate rapidly. To determine whether an otherwise undetectable cytosolic and/or ER pool might be involved
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Figure 3 Ultrastructural analysis of GM130-positive tubules and puncta with correlative light-electron microscopy (CLEM) and cryo-immunoelectromicroscopy (cryo-EM). A, B, Cells grown on cover slips with coordinated grids were fixed and labelled for GM130 in A, and for GM130 and COP in B. The cells exhibiting long GM130-positive tubular protrusions were selected and optical sections performed to determine the spatial positions of the tubules. Then cells were immunogold-labelled for GM130, embedded and 100 nm-tangential serial sections were examined without additional contrast. Aa, Low-magnification EM image of the GM130-labelled area identified by LSCM (inset). Arrowheads show reference points along the nuclear membrane. Arrows indicate the Golgi area and a tubule labelled for GM130. The rectangular box includes the GM130-positive tubule and the asterisk inside the rectangle indicates a lipid droplet. b, Enlargement of the rectangular box in a, showing the straight tangential membrane tubule (thin arrows) containing GM130 and closely associated with a lipid droplet (asterisk). The breaks along the tubule represent its bending, which is visible in consecutive sections (data not shown). c, d, Images from the same series demonstrating the GM130 localization at peripheral tubular clusters (c) and at the Golgi (d). c shows a GM130positive tubular cluster localized in an area far from the Golgi. Arrowheads indicate ER. d shows the concentration of GM130 labelling at the cis-side of the stack. B, COS7 cells were immunofluorescence-labelled for GM130 and COP. The merge of the stainings is shown in c; the single labellings of GM130 and COP are shown in a and b, respectively. d shows a low-magnification EM image of the GM130-labelled area identified by LSCM in a. The arrow in ad indicates a tubule that is positive for GM130, and also, though in a discontinous manner, for COP. e shows a larger view of box in d, including the tubular structure. Note that the tubular structure identified by immunofluorescence corresponds to a bundle of tubules with some of them containing flattened domains. C, COS7 cells were labelled with HRP-conjugated HP lectin and antiGM130 antibodies. GM130 and HP lectin colocalize and show a polarized distribution in the first two cisternae. The swelling of the cisterna is very likely due to an over-development of the diaminobenzidine reaction used for HRP detection. DF, COS7 cells were processed for cryo-EM and labelled for GM130 (10 nm gold particles, arrows) and mannosidase I (5 nm gold particles, arrowheads). GM130 and mannosidase I colocalize at the level of the first (or first two) cisterna of the Golgi stacks (D and E). Colocalization is not seen on peripheral tubular clusters that have GM130 but lack mannosidase I (E and F). Scale bars: Aa, 650 nm and inset, 2.6 m; b, 140 nm; c, 225 nm; d, 160 nm; Bac, 20 m; d, 4 m; e, 300 nm; C, 220 nm; D, 200 nm; E, 260 nm; F, 120 nm.
in GM130 dynamics, an extensive cytoplasmic area including the ER, but devoid of recognizable GFPGM130-positive structures, was bleached repeatedly (five bleaching rounds in 16 min): under these conditions no FLIP of the Golgi area was observed (Fig. 1F, b and c), indicating that a cytosolic pool and/or ER pool of GM130 does not contribute to these fast dynamic processes, and that the observed anterograde and retrograde tubules are responsible for the communication between the central and peripheral GM130 compartments. Cells that overexpress GFPGM130 (which we usually excluded from our analysis) contain a large pool of cytosolic GFPGM130. In these cells, bleaching of cytoplasmic areas devoid of GM130-positive puncta and tubules induced FLIP in the Golgi area. Given that in normal cells there is very little cytosolic GM130 (ref. 4), we assume that the cytosolic distribution of overexpressed GFPGM130 results from saturation of the membrane-binding sites (that is, GRASP65). Our results indicate that there is a fast GolgiIC recycling pathway for GM130; they do not, however, exclude the possibility that a slower recycling pathway involving the ER might exist and be relevant for longer-term processes. The peripheral GM130 tubules and puncta seem to undergo significant changes in abundance over time, even in individual cells. We observed single cells at 4-h intervals and registered the rate of puncta and tubule formation over 12 h. This rate was found to vary on average from 0.3 to 10 tubules per minute. Given the correlation between the number of GM130-positive tubules and the transport of cargo proteins (see below and Table 1), the oscillations in the abundance of puncta and tubules might reflect an undetected cyclic activity of the secretory pathway, possibly related to stages of the cell cycle. GM130-positive peripheral structures have compositional features intermediate between those of the cis-Golgi and the IC. Our observations of GM130 distribution in living cells prompted us to re-examine the distribution of endogenous GM130 and its colocalization with Golgi and IC markers by immunofluorescence. The sensitivity of the tubules to the fixatives was a technical difficulty. When cells expressing GFPGM130 were fixed with paraformaldehyde (v/v 4%), most tubules were either fragmented into strings of small punctate structures, or became extremely thin or undetectable. Nevertheless, there was a sufficient number of structures to enable us to characterize the compartment. Consistent with the observations in live cells, GM130 was localized at the central Golgi complex, at peripheral puncta, and also at the extremely thin tubules that connect the puncta with each other and with the central Golgi area (Fig. 2). Both the puncta and the tubules labelled with GM130 were devoid of bona fide Golgi markers such as giantin (Fig. 2A), the cis-Golgi markers mannosidase I and HP lectin-binding proteins25 (see below and Fig. 3), and the medial-Golgi marker mannosidase II. In contrast, there was very good colocalization of GM130 and GRASP65 under all experimental conditions (Fig. 2B), indicating that these two proteins are closely associated in the peripheral as well as in the central Golgi pool. The peripheral GM130-positive structures were detectable in different cell lines, including COS7, NRK, RBL (Fig. 2), HepG2 and HeLa cells (data not shown), although always in a fraction of the cell population (~30 10%; this estimate might be low, however, because of the transient nature of the GM130-labelled tubules and puncta and their sensitivity to fixatives). Some of the features of the GM130-positive puncta and tubules suggest a similarity with the IC10,21,26,27. To investigate, we analysed the distribution of GM130 compared with that of IC proteins, such as COPI, COP, ERGIC5326, KDEL-receptor (KDEL-R), p115 (Fig. 2B) and p23. In all cases GM130 and the IC markers colocalize (91 2% of GM130 puncta contain KDEL-R, and 78 2% of GM130 puncta contain COP), but the distribution of the IC markers is broader than that of GM130 (12 4% of KDEL-R-positive and 5 2% of COP-positive structures contain GM130). The partitioning between the Golgi and peripheral pool was different for the IC markers and GM130: about 80% (77 15%) of total
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Figure 4 GM130-positive L-IC elements contain and transport secretory cargo. COS7 cells were transfected with GFPVSV-G, kept 16 h at 40 C, shifted to 32 C for the indicated times, fixed and processed for immunofluorescence. At 40 C VSV-G is localized in the ER (data not shown). A, The cells were stained with anti-GM130 antibodies. Three minutes after the shift to 32 C (a, b), VSV-G accumulates in punctate structures (filled arrows in a), which are devoid of GM130 (empty arrows in b), whereas at 8 min after the temperature shift VSV-G colocalizes with GM130 on puncta and tubules (filled arrows in c and d). Forty minutes after the temperature shift (e, f), VSV-G localizes to the Golgi, the plasma membrane, and peripheral spots (filled arrows) that are devoid of GM130 (empty arrows) and probably represent post-Golgi transport intermediates. B, GFPVSV-G-transfected cells were double stained for KDEL-R (red) and SEC31 (blue) in a and b and for KDEL-R
(red) and GM130 (blue) in c and d. The insets show a larger view of the boxed areas in ad, and includes the single labelling and the merge of the labelling as indicated. Three minutes after the shift to 32 C (a, c), VSV-G accumulates in punctate structures that contain SEC31 (light blue spots in a) or KDEL-R (yellow spots in a and c), but that are devoid of GM130 (blue puncta in c). Note that only few VSV-G containing puncta are positive both for SEC31 and KDEL-R (white spots in a). Eight minutes after the shift to 32 C (b, d), there are still some VSV-G containing puncta positive for KDEL-R but devoid of GM130 or SEC31 (yellow spots in b and d) but also VSV-G containing puncta positive both for KDEL and GM130 (white spots and tubule in d). Note that at this time only few VSV-G puncta contain SEC31 (light blue puncta in b). Scale bars, A, 10 m; B, 4 m.
GM130 labelling but only 40% (41 12%) of total COPI labelling coincided with the central Golgi area. All the non-Golgi GM130 labelling fell in the next 10 m around the Golgi area (the average diameter of COS7 cells is around 30 m), whereas only 60% of the non-Golgi COPI labelling was present in the same area, with the rest distributed in more peripheral zones. Also, the average size of COPI elements was smaller than those of GM130 puncta (the apparent average diameter of COPI-positive and GM130-negative elements was 0.25 m and of COPI- and GM130-positive elements was 0.6 m). We also analysed the extent of colocalization of GM130 with the IC markers on the tubules. We found that it was higher (80 7%) in the case of proteins such as KDEL-R and p23 that cycle between the Golgi and the ER than in the case of those such as ERGIC53 that cycle mostly in peripheral compartments
(that is, between the IC and the ER (30 7%)). COPI and p115 were not uniformly distributed along the GM130-positive tubules, but were organized in spots collinear with the tubules (Fig. 2B). These data indicate that GM130 delineates a subdomain of the IC, which includes the larger IC elements that are distributed in a wide area (with an average radius of 10 m) around the Golgi complex, and communicate with it via anterograde and retrograde tubules. There was poor colocalization between GM130 and proteins localized at the ERES, such as the COPII components SEC13 or SEC31 (less than 10% of GM130 puncta colocalized with SEC31; Fig. 2B). There was only a partial overlap between the ERES markers and the IC markers (KDEL-R): 17 4% of KDEL-positive elements apparently colocalized with SEC31 and 16 5% of SEC31
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Figure 5 GM130-positive L-IC elements transport secretory cargo. a, b, Dynamics of VSV-G transport to and from the GM130-positive tubules and L-IC stations. COS7 cells doubly transfected with CFPVSV-G (green) and YFPGM130 (red) were kept overnight at 40 C and then shifted to 32 C. a and b show individuals frames of movies taken 1015 min after release from the 40 C block. a, A GM130-positive puncta (L-IC, filled arrowhead) initially devoid of VSV-G (empty arrowhead), acquires VSV-G (00.04), and generates/transforms into a tubule (00.06). Afterwards, the tubule reaches (00.28) and disappears into the central Golgi area (00.32). b, A GM130- and VSV-G-positive tubule (filled arrowhead) originates from a GM130- and VSV-G-positive puncta (00.00), detaches from the puncta (00.02), moves (00.11, 00.17), then reaches and fuses with a neighbouring puncta (00.21). Scale bars, 2 m. c, VSV-infected COS7 cells were kept for 2 h at 40 C and then shifted to 32 C for 8 min. The cells were processed for cryo-EM and the sections were double stained for GM130 (5 nm gold particles, arrowheads) and VSV-G (10 nm gold particles, arrows). Note that GM130 and VSV-G colocalize at the cis-cisterna and at vesicular and tubular profiles juxtaposed to the cis-cisternae. Scale bar, 70 nm.
elements colocalized with KDEL. It is worth noting that the stainings of SEC31 and KDEL confirmed previous observations22, indicating that the labelled structures might correspond to subdomains of the same structure overlapped. Finally, in triple-labelling experiments, it was possible to follow the distribution of an ERES marker (SEC31), an IC marker (ERGIC53) and GM130 in the same cell (Fig. 2C). Without taking into account the central Golgi area where, due to the resolution limits of the confocal analysis, it was impossible to distinguish specific
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staining patterns, four main populations were identified: one labelled exclusively by SEC31 (blue spots in Fig. 2C), one by SEC31 and ERGIC53 (purple spots, which often show a local segregation of red and blue staining), one labelled exclusively by ERGIC53 (red spots), and one, representing the GM130-positive subdomain of the IC, labelled by ERGIC53 and GM130 (yellow spots and tubules). Ultrastructure of the peripheral IC elements containing GM130. We next characterized the ultrastructure of the puncta and bona fide tubules positive for GM130 using correlative light-electron microscopy (CLEM28,29). This technique allows the ultrastructure of individual cells and organelles previously identified at the immunofluorescence level to be analysed (Fig. 3A and B). In the central Golgi area, GM130 was mainly restricted to the cis face of the Golgi4, where it localized to the first two cisternae of the stacks and colocalized with the HP lectin25 (Fig. 3C). The GM130 label was present also on a variety of complex membranes comprising: flat reticulated cisterna-like structures underlying the first cisterna of the stacks; tubulo-reticular elements apparently connecting adjacent stacks (presumably belonging to the noncompact zone); and clusters of convoluted tubular profiles (with an average diameter of 800 nm). The tubular clusters were identified as typical IC elements after analysis of serial sections. These labelled IC membranes were located not only close to the Golgi area but also in areas distant from the Golgi (Fig. 3Ac). These peripheral elements corresponded to the large GM130-positive puncta observed in immunofluorescence experiments (see above). GM130 antibodies also labelled long and continous membranous tubules (defined as tubular profiles visible in no more than two consecutive 100-nm-thick serial sections) emanating from GM130-stained tubular clusters. These tubules were 50100 nm thick and 312 m long (Fig. 3A and B). Usually the tubules were straight or slightly bent. Strikingly, many were arranged in pairs or in bundles with some of them containing flattened domains (Fig. 3B), and in these cases the tubules corresponded at the immunofluorescence level to thick tubular structures containing both GM130 and COPI. Using cryoimmunoelectron microscopy (cryo-EM), we found that mannosidase I colocalized with GM130 on the cis-Golgi cisterna, but was not present on the peripheral tubular clusters containing GM130 (Fig. 3DF). The EM results described above were consistent with observations both in living and in fixed cells, and showed that GM130 associates not only with the cis-Golgi cisternae and tubular clusters close to the Golgi stacks, but also with peripheral straight tubules and tubular clusters containing IC markers and devoid of cis-Golgi markers. GM130-positive IC elements carry secretory cargo. To assess whether the IC elements that contain GM130 are involved in transporting cargo, the distribution of GM130 was studied simultaneously with that of a secretory membrane protein, the temperaturesensitive variant of the glycoprotein (G) from the ts045 strain of the vesicular stomatitis virus (VSV). Transport of the VSV-G protein from the ER to the Golgi can be synchronized because it folds incorrectly and is retained in the ER at 40 C; correct folding and release occurs after shifting to 32 C. Cells were transfected with GFPVSV-G, kept 16 h at 40 C to accumulate the VSV-G in the ER, shifted to 32 C for different time intervals, and then processed for immunofluorescence and stained for GM130 (Fig. 4A) or for SEC31 and KDEL-R or for KDEL-R and GM130 (Fig. 4B). Three minutes after the shift to 32 C VSV-G accumulates in punctate structures devoid of GM130, and at 8 min VSV-G colocalizes with GM130 on puncta and tubules (Fig. 4A). The punctate structures that contain VSV-G at early times (3 min) show very good colocalization with the ERES marker SEC31 (58% of VSV-G positive elements contained SEC31) and with the IC markers KDEL-R (87% of VSV-G positive elements contained KDEL-R). We were able to distinguish a population of VSV-G-containing structures positive for IC markers, but not for SEC31 (Fig. 4B, a). At these times (23 min) the colocalization between VSV-G and GM130 was poor
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Figure 6 GM130 distribution after synchronization at 15 C. A, VSV-infected COS7 cells (ac) were kept for 2 h at 40 C and then shifted for an additional 2 h at 15 C. The cells were double stained with anti-GM130 (a) and anti-VSV-G (b) antibodies; the merged pattern is shown in c. Note that the more central and larger puncta positive for VSV-G also contain GM130 (filled arrowheads), whereas the more peripheral puncta contain VSV-G (filled arrows) but are devoid of GM130 (empty arrows). COS7 cells (df, jl) or NRK cells (gi) were incubated for 2 h at 15 C, processed for immunofluorescence and then double stained for GM130 (d, g, j) and ERGIC53 (e), KDEL-R (h) or SEC13 (k). The merges of the two staining patterns are shown in f, i, and l. Note that GM130 partially redistributes to peripheral puncta, there colocalizing with markers of the IC (ERGIC53, KDEL-R, filled arrowheads). However, the IC markers are also present on more peripheral spots (filled arrows) devoid of GM130 (empty arrows). Note that in contrast to what is observed under steady-state conditions shown in Fig. 2, there is a marginal colocalization between GM130 and SEC-13 on punctate structures (filled arrowheads); see
text for details. mo show the transport of GM130 after release from the 15 C block. VSV-infected COS7 cells, were kept for 2 h at 40 C, for an additional 2 h at 15 C and then shifted to 32 C for 3 min. The cells were fixed, processed for immunofluorescence and stained for GM130 and VSV-G. Note that a large number of tubules containing GM130 and VSV-G (filled arrowheads) connect the peripheral puncta with the central Golgi area. B, Dynamics of YFPGM130 and CFP-VSV-G upon release from the 15 C block. COS7 cells double-transfected with YFPGM130 and CFPVSV-G were kept overnight at 40 C, for additional 2 h at 15 C and then shifted at 32 C and imaged. After 4 min at 32 C (00.30) the central Golgi area (dotted line) was bleached (00.00) by 200 iterations at maximal laser-beam intensity and the cells were imaged for an additional 2.5 min. Note that many tubules containing VSV-G and GM130 (filled arrowheads) move towards the central Golgi area, and that the latter gradually reacquires both VSV-G and GM130fluorescence. Scale bars, 10 m.
(less than 10% of GM130-positive puncta contained VSV-G; Fig. 4A, d, and B, c). After 510 min the VSV-G became associated with larger spots and long tubules connecting the spots to each other and to the central Golgi area. At this stage, many (85 10%) GM130-positive structures were positive for VSV-G (Fig. 4A and B, d). At later times (15 min), VSV-G concentrated into the central Golgi area, and 3040 min after the temperature shift, VSV-G localized on both puncta and tubules (probably representing post-Golgi transport intermediates), which were not labelled for either IC markers or GM130, and on the plasma membrane (Fig. 4A, c and f). We define as early-IC (E-IC) elements that receive cargo soon (23 min) after warming to 32 C. E-IC are mainly peripheral and contain IC markers but are devoid of COPII and GM130 (yellow spots in Fig. 4B, c and d). We define as late-IC elements that receive cargo 510 min after warming to 32 C. These elements are less peripheral than the E-IC, and contain both GM130 and IC markers (white spots in Fig. 4B, d), but are devoid of cis-Golgi markers (such as mannosidase I or HP; Figs 1A and 3E).
We examined the progression of VSV-G through the pre-Golgi transport segment using time-lapse imaging in living cells expressing CFPVSV-G and YFPGM130 (Fig. 5A). After 16 h of incubation at 40 C, VSV-G was found in the ER, whereas GM130 was associated with the Golgi complex, as well as with peripheral puncta and tubules. After shifting to 32 C, VSV-G progressively associated with punctate structures that moved centripetally in a stop-and-go fashion, as described previously19,20. Consistent with the immunofluorescence data reported above, the extent of colocalization between GM130 and VSV-G was low when the cargo protein was in the small punctate peripheral structures (that is, ERES and E-IC elements), and increased when the cargo reached the larger and more central elements of the IC (that is, L-IC elements). Indeed, many of the puncta that contained GM130 were initially devoid of VSV-G, but acquired it later. The transfer of cargo to the L-IC units occurred mainly by direct contact between VSV-G-containing E-IC elements and GM130-positive tubules and/or puncta. After cargo receipt, the GM130 puncta behaved dynamically in several ways. Some transformed into tubules that extended and moved to the central Golgi,
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Figure 7 p115 and GRASP65 control the dynamics of GM130-positive L-IC stations and tubules. A, Dynamics of GFPG95 in living COS7 cells (see also Supplementary information movie 4). Individual images from a movie of a cell expressing GFPG95, a truncated form of GM130 unable to bind p115. Time is given in min.sec. Individual spots and tubules are indicated with filled arrowheads. Note the abundance of peripheral structures containing GFPG95. Frames 00.0800.16 show a tubule originating from a peripheral L-IC, moving towards, and fusing with another peripheral L-IC. Frames 00.4001.15 show a tubule originating from the Golgi area, moving retrogradely and becomes a peripheral L-IC station. B, G95 induces the tubular transformation of the IC. COS7 cells were transfected with FlagG95 and double-stained with anti-Flag antibodies (a, c) and anti-GM130 (b) or
anti-p23 (d) antibodies. Note the tubular staining pattern of GM130 and p23 in cells expressing FlagG95 as compared with control cells. C, Masking the GM130-binding site of p115 with GM130/300N (a truncated form of GM130 that can bind p115, but not GRASP65) redistributes p115 from the Golgi to the E-IC elements. COS7 cells were transfected with FlagGM130/300N and double stained with antiFlag (a) and anti-p115 (b) antibodies. Note that GM130/300N does not localize to the Golgi or central L-IC but on peripheral puncta (two of which are indicated with filled arrowheads), there colocalizing with p115. Also, in cells expressing GM130/300N, p115 is redistributed to the periphery (b), suggesting that the docking/fusion of the E-IC with the more central L-ICs, and/or the Golgi area, is inhibited. Scale bars, A, 10 m; B, 20 m; C, 12 m.
and then disappeared into it (Fig. 5A). Sometimes cargo-containing tubules detached from the GM130- and VSV-G-positive L-IC stations, moved towards the Golgi area or other GM130-positive elements, and finally fused with them (Fig. 5B). Less frequently, GM130-positive tubules emanated from the L-IC stations and established stable (or repetitive) connections between these stations and the Golgi; in these cases, VSV-G-positive spots could be seen moving along/within the GM130-positive tubules. Finally, the cargo and GM130-containing L-IC stations established multiple, variably transient tubular connections with other stations and with the central Golgi area and underwent homotypic fusion. These observations indicate that the GM130-positive L-IC receives the ER-derived cargo from earlier IC elements and delivers it to the Golgi complex via anterograde tubules. An apparent discrepancy with this conclusion was that in a few cases the cargo appeared to arrive directly at the central Golgi area (inside a GM130-negative EIC element), apparently bypassing the GM130-positive L-IC and tubules. However, and as is shown by the EM data in Fig. 3, there are numerous GM130-containing tubular clusters in the central Golgi area. These exceptions might, therefore, be due to the resolution limits of the LSCM analysis, which allows the arrival of cargocontaining E-IC elements at the L-IC stations to be detected only when these stations are sufficiently far from the Golgi complex (and not when the L-IC stations are very close to, and not resolvable from, the central Golgi). We therefore used cryo-EM studies to
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examine VSV-infected cells: these studies showed a high degree of colocalization of VSV-G with GM130 at the level of central tubular clusters (Fig. 5C). The same studies showed that not all the peripheral tubular clusters containing VSV-G contained GM130, confirming the observation that the GM130-positive tubular clusters identify a subdomain of the IC. Finally, we studied the dynamics of GM130-positive L-IC and tubules under conditions in which the ERGolgi membrane transport is arrested at the IC by a prolonged exposure to 15 C (refs 10, 17, 21, 26, 27; Fig. 6). After 2 h at 15 C, the IC of VSV-infected cells was filled with VSV-G and, consistent with previously published reports30, was enlarged compared with that of noninfected cells. Under these conditions, the association of GM130 with the IC was increased (60% of cells had peripheral GM130-labelled puncta in addition to the central Golgi staining, compared with 30% under control conditions), confirming that the protein undergoes continuous and fast recycling through the IC. Qualitatively, however, the situation was similar to that observed at 3237 C. Also at 15 C, only a subpopulation of the IC elements was filled with VSV-G, including the less peripheral and larger stations (the L-IC), and were positive for GM130 (Fig. 6A). At the same time, the colocalization of the recycling proteins KDEL-R and ERGIC53 with GM130 remained partial at 15 C. KDEL-R and ERGIC53 were present in the small, very peripheral spots, as well as in larger, more central spots; GM130 was exclusively in the latter (Fig. 6A). The
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Figure 8. Interfering with GM130-tubule dynamics with G95 inhibits the transport of VSV-G protein to the Golgi complex. A, COS7 cells were transfected with GFPG95. After overnight expression, the cells were infected with VSV, incubated at 40 C for 2 h and shifted to 32 C for 15 min (ac), or incubated at 40 C for 2 h, at 15 C for an additional 2 h, and then shifted to 32 C for 5 min (df). The cells were processed for immunofluorescence and labelled with anti-VSVG (b, e) or anti-giantin (c, f) antibodies. Note that 15 min (b) and 5 min (e) after the shift to 32 C, the VSV-G staining pattern is tubular and/or punctate in G95-transfected cells whereas it is mainly central in control cells, indicating a delay in VSV-G arrival to the Golgi complex. Note that the general organization of the Golgi complex, as assessed by giantin staining (c, f) is not perturbed in cells expressing G95 at low-to-intermediate levels. Scale bars, 20 m. B, G95 inhibits the transport of VSV-G from L-IC to the Golgi complex. COS7 cells transfected with GFPG95 (G95) or with GFP alone (control) were infected with VSV, incubated at 40 C for 2 h and shifted to 32 C for the indicated times (a), or incubated at 40 C for 2 h, at 15 C for an additional 2 h, and then shifted to 32 C for the indicated times (b). The cells were fixed, processed for immunofluorescence and stained with anti-VSV-G anti-
body. At each time point, 200 cells were analysed for the VSV-G staining pattern and classified as having a punctate pattern when they possessed more than five (in a) or ten (in b) peripheral structures (tubules or puncta) containing VSV-G. The results are expressed as percentage of cells exhibiting a punctate pattern of VSV-G staining and are the means (SD) of three independent experiments. Note that at time 0 in a very few cells, if any, presented a punctate pattern, whereas in b almost every cell had a punctate pattern. At late times, few cells had puncta. These puncta were devoid of GM130, and probably represented post-Golgi transport intermediates. C, The L-IC is a subdomain of the IC, characterized by the presence of IC markers and the cis-Golgi matrix proteins GM130 and GRASP65, by the absence of the cis-Golgi markers mannosidase I and HP lectin, and by the propensity to generate tubules that connect L-IC units to each other and to the Golgi. The transition from E-IC (containing IC markers but not GM130 and GRASP65) to L-IC (dotted circles) occurs mainly by fusion of E-IC with GM130-positive tubules (a) or L-IC stations (b). The incorporation of L-IC into the Golgi complex (rectangles), might involve one (or both) of two mechanisms: either fusion of L-IC with the cis-Golgi cisterna (c), or juxtaposition and flattening of L-IC under the cis-most cisterna (d).
only difference was that GM130, which does not colocalize at the periphery with the ERES marker SEC13 at 37 C, did colocalize with SEC13 at 15 C, although partially and only at the level of the larger SEC13-positive units. These findings are consistent with the recent observation that the ERES and the IC elements, which are distinguishable at 37 C, can mix at 15 C (ref. 22). When the cells previously incubated at 15 C were warmed (to 32 C or 37 C), there was a massive proliferation of the GM130positive tubules starting from the peripheral puncta. Most tubules
contained VSV-G (Fig. 6A, mo) and anterogradely transported both GM130 and VSV-G to the Golgi complex, as indicated by the fast refilling from peripheral puncta of the central Golgi area after its photobleaching (Fig. 6B; and Supplementary Information movie 3). In addition, these GM130 tubules did not colocalize with ERGIC53-labelled tubules that also proliferate after removal of the 15 C block and, in contrast, were mainly retrograde and directed to the ER (see also refs 10, 26). This distinct behaviour of the two recycling proteins GM130 and ERGIC53 suggests that they follow
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different recycling pathways and that the GM130 recycling loop is shorter than that of ERGIC53 and operates between the Golgi and L-IC. Interestingly, the L-IC was more developed and the GM130 tubulation was more pronounced (both in terms of number of GM130-positive tubules per cell and number of cells presenting GM130-positive tubules) in VSV-infected cells compared with control cells (or VSV-infected cells treated with cycloheximide; Table 1 and Fig. 4A, e) under temperature conditions that are permissive for the ER-to-Golgi transport of VSV-G. These results are consistent with the possibility that the abundance of the L-IC might reflect the activity rate of the secretory pathway, and suggest that tubulation of GM130 might be controlled by the presence of cargo inside the IC. Molecular interactions regulating GM130 recycling between the Golgi complex and the L-IC. The main proteins that interact with GM130 are GRASP65 and p115. GRASP65 is tightly associated with the Golgi membranes, forms a very stable complex with GM130, and is responsible for GM130 association with Golgi membranes6. p115 was originally identified as a protein involved in intra-Golgi transport and transcytosis31,32, associates with GM130 and other matrix proteins (such as giantin, and golgin160; P.M. and M.A.D.M., unpublished observations), is localized on the Golgi complex and at the IC, and participates, together with GM130 and GRASP65, in tethering complexes thought to function in membrane transport and in cisternae stacking at the Golgi-complex level7,8,33,34. We investigated whether the interaction of GM130 with these proteins controls the dynamics of the GM130-containing LIC. Because we know that GM130 interacts with GRASP65 and p115 via its carboxy and amino termini, respectively35,36, we constructed protein mutants that would disrupt these interactions. First, we studied the distribution of a truncated form of GM130, G95, that lacks the p115-binding domain, but contains the GRASP65-binding domain. GFPG95 localized mainly on peripheral tubules, but also at the central Golgi (Fig. 7A). The GFPG95 tubules were extremely dynamic and moved and extended with a speed similar to that of GFPGM130 tubules (Fig. 7A and Supplementary Information movie 4). However, when compared with GM130 tubules, they persisted longer (their average lifespan was 14 s compared with 5 s for GM130 tubules). The G95 tubules also showed more numerous and random changes in direction before projecting towards, contacting, and eventually disappearing into, other puncta or the central Golgi area. In fact, the G95 tubules had prevailing lateral direction, and a less obvious anterograde direction when compared with the GM130 tubules (Fig. 7A; and Supplementary Information movie 4). The above pattern was not exclusive to GFPG95 but was also observed with other G95 constructs such as FlagG95 (Fig. 7B). One explanation for the effect of G95 might be the inability of the truncated G95 protein to bind p115, and thus mediate the effective docking of the tubules to the Golgi complex. The fact that the block was not complete could be due to the inability of G95 to completely inhibit the action of the endogenous nontruncated GM130 protein, or redundant docking mechanisms. We next studied the distribution of a truncated form of GM130, GM130/300N, which lacks the GRASP65-binding site but contains the p115-binding site. GM130/300N did not localize to the central Golgi or to the tubules, indicating that GRASP65 mediates the association of GM130 with Golgi6 and pre-Golgi membranes (Fig. 7C). In fact, probably because of its ability to bind p115, GM130/300N colocalized with p115 on peripheral elements of the IC (E-IC), with which p115 associates in a GM130-independent way37. Interestingly, GM130/300N caused a redistribution of p115 from the Golgi area to the peripheral E-IC elements (Fig. 7C). These results indicate that interfering with the interaction of p115 with endogenous GM130 (the distribution of which at the Golgi and LIC was unchanged by GM130/300N, data not shown) interferes with the normal p115 cycling between the Golgi and the periphery.
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It has been recently proposed that p115 cycles through pre-Golgi compartments in an exclusively membrane-associated form38 and is not released into a cytosolic pool. Therefore, interfering with the interaction of p115 with GM130 inhibits the progression of p115positive E-IC to later and more central stations and suggests that the docking of E-IC with L-IC elements (both tubules and stations) might be controlled by p115. Another truncated form of GM130, G95/GRASP65, which lacks both the p115- and GRASP65-binding sites, is completely distributed in the cytosol (data not shown), confirming that GRASP65 mediates the association of GM130 with the Golgi and pre-Golgi membranes. Together these results indicate that the interaction between GM130 and p115 is required for the anterograde transport and docking of L-IC elements to the central Golgi and suggest that it might also control the docking of E-IC to L-IC elements. GM130-positive tubules mediate the incorporation of L-ICs and cargo into the Golgi complex. We next investigated how the GM130p115 interaction functions in ER-to-Golgi transport. We analysed the impact of G95, a truncated GM130, on the transport of neosynthesized VSV-G to the Golgi complex (Fig. 8). The secretion of VSV-G from the ER and its arrival to the E-IC and L-IC were not affected, whereas its arrival at the Golgi complex was inhibited in G95-transfected cells, as compared with control cells (either nontransfected cells or cells transfected with GFPG95/GRASP65 or GFP alone). After 15 min at 32 C in control cells, VSV-G was mainly concentrated in the Golgi; only 25% of cells presented a consistent fraction of VSV-G in peripheral puncta, in contrast to 84 15% of the cells transfected with G95, in which VSV-G was mainly present in peripheral puncta and tubules with lower Golgi staining (Fig. 8A, ac). This different distribution was maintained at later time points (45 min) when 55 8% of G95-transfected cells and 15 3% of control cells presented VSVG-positive peripheral puncta and tubules, indicating a defect in the transport to the Golgi system (Fig. 8B, a). Similarly, in cells kept at 15 C for 2 h and then shifted to 32 C, the G95-transfected cells had a slower rate of transport of VSV-G to the Golgi compared with control cells (Fig. 8A, df). Under these conditions after 5 min at 32 C, 86 10% of G95-transfected cells had a large proportion of VSV-G still in the peripheral puncta, whereas in 88 9% of control cells the main fraction of VSV-G was in the central Golgi area (Fig. 8B, b). This difference in VSV-G distribution was maximal after 5 and 12 min at 32 C and decreased at later time points (30 min). Interfering with the dynamics of GM130 tubules impairs the IC-to-Golgi transport of cargo. G95 tubules have a longer half-life compared with GM130 tubules, and are defective in docking and fusing with the Golgi complex. This docking defect might explain why the cargo is not efficiently delivered from the L-IC to the central Golgi complex.
Discussion
The main finding in this report is that the cis-Golgi proteins GM130 and GRASP65 cycle via membrane tubules through specialized stations of the IC, the L-IC. This L-IC does not contain cisGolgi markers (such as mannosidase I and HP lectin binding proteins) and contains, at any given time, only a relatively minor portion (20%) of GM130 and GRASP65, with the rest of the proteins remaining associated with the central Golgi complex. However, the two pools of GM130 are not functionally separated, rather, they exchange rapidly, as indicated by the fluorescence loss in the peripheral pool induced by bleaching the central pool and vice versa, and by the fast recovery of the fluorescence of the central and peripheral pool after bleaching. The L-IC represents a subdomain of the IC where the transition between the IC and Golgi occurs. L-IC stations are present in the central Golgi area as well as in the cell periphery. Although
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physically separated, they are interconnected by membranous tubules establishing functional and often physical continuity among the various central and peripheral stations. As a result, the L-IC stations probably function as a homogeneous, well-mixed compartment (Fig. 8C). Morphologically and compositionally, the L-IC stations are similar to traditional IC elements in that they exhibit the typical IC conformation of convoluted tubular clusters, and contain IC markers. However, they also differ from the E-IC elements, by the presence of the matrix proteins GM130 and GRASP65, by having a more central location, and a different role in the progression of cargo towards the central Golgi. This is apparent as they receive ER-derived cargo with a delay (of 34 min) from its appearance in the ERES and E-IC and before its arrival at the central Golgi complex. The transfer of cargo from E-IC to L-IC occurs mainly through a direct fusion of E-IC elements with GM130-positive tubules or a GM130-positive L-IC stations (Fig. 8C, a and b). However, we cannot at present exclude a further possibility, that the transfer from E-IC to L-IC might occur by a dissociative process in which cargo-containing carriers detach from E-IC and are transported to the L-IC. The L-IC is thus analogous to late endosomes, which receive cargo from early endosomes and send it to lysosomes or to the Golgi. Our working model is that cargo must be first incorporated into the GM130-containing L-IC before entering the Golgi stack. This entry step is a critical transition in the secretory process. Because the flat, reticulated, cisterna-like structures of the L-IC contain GM130, we believe that its formation represents the next step in the entry process. This formation might involve one of two alternative mechanisms: either the fusion of L-IC stations with themselves and the cis-Golgi cisterna, followed by flattening of the membranes; or simply juxtaposition and flattening of L-IC stations under the cis-most cisterna (Fig. 8C, c and d). It is logical to think that, in their capacity as Golgi matrix proteins, GM130 and GRASP65 might participate in the flattening and the morphogenesis of this cis structure. The availability of GM130 and GRASP65 as markers of the interface between the IC and the Golgi should facilitate the dissection of these critical events. The carriers that operate in the late transport steps from the IC to the Golgi are either the L-IC stations themselves moving en bloc towards their destinations, or the membranous tubules emanating from these stations (Fig. 8). The abundance and rapid turnover of these tubules suggests they have a major role. They reach their destinations in several ways: they can dissociate from the original L-IC station and dock and fuse with the Golgi; they can move en bloc together with the L-IC station towards the Golgi complex and subsequenly fuse with the Golgi membranes; or they can form more stable connections between the L-IC station and the central Golgi in which the cargo appear to move as a bolus39. A recent study describes tubular structures emanating from IC elements (after release from a 15 C block) that have been proposed as specific carriers for soluble cargo; membrane proteins like VSV-G appear in these tubular structures only when the proteins are heavily overexpressed40. These observations differ from ours as we do see the membrane protein VSV-G in GM130-positive tubules even at very low levels of expression and/or infection. It is possible that the soluble GFP-containing tubules described previously40 and the VSVG- and GM130-containing tubules described here represent two separate transport systems. We also found that the association of GM130 with soluble GFP-containing tubules is poor (data not shown), which is consistent with two transport systems. Other evidence supports the idea of a major function of the tubules in transport. Interfering with the docking of the tubules to the Golgi complex with the truncated form of GM130, G95, slows down the anterograde transport of VSV-G from the L-IC stations to the Golgi complex. Our results with G95, which indicate that GM130 is involved in ER-to-Golgi transport in intact and living cells, are in line with recent data obtained in semi-intact cells using anti-GM130 antibodies33,41. The transport block with G95 is partial, however. This might be due to the fact that G95 does not completely inhibit the
action of the endogenous, nontruncated, GM130 protein, or to the existence of redundant docking mechanisms. That tubules increase both in terms of number of tubules per cell and of number of cells with tubules when the demand for anterograde transport increases (for example, after release of the 15 C block, when the cargo that has accumulated in the IC has to be transported to the Golgi complex) is also consistent with the L-IC stations and tubules being involved in membrane transport to the Golgi complex. Remarkably, the extent of GM130 tubulation correlates with the extent of cargo transport, and seems to be controlled by the amount of cargo inside the IC the tubulation is more evident in VSV-infected cells compared with noninfected cells, and it can be partially prevented by protein synthesis inhibitors. The mechanisms and sensors underlying this regulation have not been identified. What are the relationships that exist between the GM130-positive L-IC tubule system and the molecular machineries known to operate in ER-to-Golgi transport (involving COPII, COPI, SNAREs, rab1 and p115)? The GM130-positive L-IC stations contain COPI (but not COPII) components, which are patchily distributed along the L-IC-associated tubules. As for the SNARE proteins, it is worth noting that the localization and dynamics of GM130 resemble those described for membrin42,43. Unlike other SNAREs involved in ER-toGolgi transport, such as rbet1 and sec22, membrin is enriched in mature or Golgi-adjacent IC elements, and cycles between these and the cis-Golgi42,43. Membrin is thus a good candidate for the control of docking/fusion events involving L-IC stations and tubules. Finally, the recently reported ability of rab1 to interact with GM130 (refs 41, 44) raises the intriguing possibility that the dynamics and the function of GM130 might be controlled by this small GTPase. p115 plays an important role in ER-to-Golgi transport33,37. It is recruited to COPII vesicles and interacts with selected SNAREs; this interaction is controlled by the small GTPase rab1 (ref. 45). By using the truncated forms of GM130 lacking the p115- or GRASP65binding domains, we have shown that p115 controls two distinct steps of the transport of GM130-positive, L-IC elements the docking (and fusion) of L-IC elements to the central Golgi and probably also the docking of p115-containing E-IC elements to the GM130-containing L-IC stations. We have identified a specialized domain of the IC, the L-IC, where important maturative events of ER-derived membranes occur, such as the acquisition of structural Golgi components and the homotypic fusion between L-IC units and their interconnection via membrane tubules. These biochemical and morphological changes may be important in driving the process of incorporation of neosynthesized membranes into Golgi membranes. The definition of the molecular mechanisms involved in this GM130-regulated tubulation process, the understanding of the regulation of GM130 dynamics, and the identification of its molecular partners on the tubules and at the L-IC stations remain challenges for future studies.
Methods
Reagents and antibodies
All chemical reagents were of analytical grade or higher and purchased from Sigma (Saint Louis, MO) unless otherwise specified. Cell-culture media were obtained from Gibco (Grand Island, NY). Two distinct polyclonal anti-G95 antibodies and the polyclonal anti-giantin antibody were prepared as described previously46. The polyclonal anti-G95 antibody was affinity purified on GSTG95 immobilized on a glutathione-Sepharose 4B column (Amersham Biosciences, Buckinghamshire, UK). Antip115 polyclonal antibodies were generated by immunizing against a fusion protein between glutathione S-transferase (GST) and a C-terminal fragment of p115 (GSTp115-600C), and affinity purified on p115-600C. The anti-GM130 and anti-KDEL-R monoclonal antibodies were from Transduction Laboratories (Lexington, KY) and from StressGen Biotechnologies (Victoria, Canada), respectively. The monoclonal anti-VSV-G clone P5D4 Cy3-conjugated and the anti-Flagbiotinylated M5 monoclonal antibodies were from Sigma (Saint Louis, MO). The polyclonal anti-mannosidase II and anti-mannosidase IA was provided by K. W. Moremen (University of Georgia, USA). The polyclonal anti-SEC13 and anti-SEC31 antibodies were provided by B. L. Tang (University of Singapore). The polyclonal anti-p23 antibody was provided by J. Gruenberg (University of Geneva, Switzerland). The polyclonal anti-COP antibody was provided by J. Lippincott-Schwartz (National Institutes of Health, Bethesda, MD). The polyclonal anti-GRASP65 antibody was provided by F. Barr (Max Planck
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Table 1 GM130-positive tubule proliferation during IC-to Golgi transport ofsecretory cargo
above, were incubated with GSTp115-600C immobilized on a glutathione Sepharose column, for 1 h at 4 C, and, after extensive washes in PBS, the retained proteins were eluted with 1 M glycine and analysed by SDSPAGE. After electroblotting, the nitrocellulose filters were incubated with antiGM130 antibodies and then subjected to chemiluminescence detection. The proteins of interest (that is, the bands corresponding to the endogenous GM130 (relative molecular mass 130,000; Mr 130K), and the recombinant GFPGM130 protein (Mr 160K) were quantified by densitometry1, and the coprecipitated or retained proteins were expressed as percentages of the total protein present in the extract. In the immunoprecipitation experiments, 14 3% of the total endogenous GM130 protein and 12 4% of the total recombinant GFPGM130 protein (which was 34-fold more abundant than endogenous GM130) was coimmunoprecipitated with p115. The efficiency of p115 immunoprecipitation in the different samples was comparable as assessed by testing the immunoprecipitates for the presence of p115. In the p115-600C affinity column experiments, 12 3% of the endogenous GM130 and 10 2% of the recombinant GFPGM130 present in the lysates were specifically retained. Truncated human GM130 constructs. The G95 constructs46, encoding amino acids 371990, were obtained by subcloning G95 into the pEGFP-C1 vector and into the pcDNAIII1AB vector. The recombinant protein was able to bind GRASP65 but not p115, as assessed in immunoprecipitation studies performed in transfected COS7 cells (see above). The G95/GRASP65 construct, which encodes residues 371961, was generated by removing the SmaI fragment from the pEGFP-C1/G95 vector. The GM130/300N constructs, which encode residues 1307, were generated by in-frame insertion of the HindIII cDNA fragment of pEYFP-C2/GM130 into either the HindIII restriction site of the pEGFP-C2 or the HindIII restriction site of the pcDNAIII 1AB. CFPVSV-G. The cDNA covering the entire coding region of the temperature-sensitive VSV-G protein47 was inserted into the EcoRI/SalI-digested pECFP-N1 plasmid (Clontech).
Percentage tubulated cells 15 C Exposed to 32 C after 15 C Non-infected 11 3 cells (3 1) VSV-infected 14 2 cells (4 1) VSV-infected 12 1 cells + CHX (4 1) 25 3 (18 2) 60 5 (48 4) 17 2 (6 2) 20 3 (9 1) 29 2 (8 2) 22 3 (3 1) 40 C Exposed to 32 C after 40 C 22 4 (14 2) 59 4 (44 3) 30 3 (4 1)
COS7 cells were infected (or not) with VSV, were incubated for 2 h at 40 C and then shifted to 32 C for 10 min. Alternatively they were incubated for 2 h at 40 C, kept for additional 2 h at 15 C and then shifted to 32 C for 3 min. Where indicated, VSV-infected cells subjected to the described temperature shifts were treated with 100 g ml1 cycloheximide (+ CHX) at the beginning of the 40 C incubation period. At the end of each incubation treatment, the cells were fixed, processed for immunofluorescence, and stained with anti-GM130 (and anti-VSV-G) antibodies, and the number of tubules per cell was quantified. At least 200 cells for each treatment were counted and classified as tubulated (at least one GM130-positive tubule) or highly tubulated (more than three GM130-positive tubules; these values are shown in parentheses). Experiments were carried out in triplicate; results are means s.d.
Immunofluorescence analyses
COS7 and NRK cells grown on glass coverslips were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and processed for immunofluorescence as described previously2. The HP staining was performed in accordance with ref. 48. In selected experiments, COS7 cells were treated with extraction buffer (100 mM MES, pH 6.5, 1 mM EGTA, 0.5 mM MgCl2, 1 mM NaN3, and protease inhibitors) for 10 min at room temperature. Analysis of VSV-G protein transport was performed in cells infected with the ts045 VSV strain or transfected with GFPVSV-G. COS7 and NRK cells, plated on coverslips, were infected with VSV at 32 C for 45 min, incubated at 40 C for 2 h, and then either shifted to 32 C for different times or further incubated at 15 C for 2 h (to permit accumulation of the protein in the IC) before being shifted to 32 C for different times. At the end of the incubations, the cells were fixed and processed for immunofluorescence. Quantification of VSV-G transport was performed by analysing the immunostaining patterns of at least 200 cells in three separate experiments. The results are given in the figure legends or in the text, as percentage (s.d.) of cells presenting a given pattern. For confocal imaging, samples were examinated with a ZEISS LSM 510 confocal microscope. Optical sections were obtained under a 63 oil-immersion objective, at a definition of 1,024 1,024 pixels, adjusting the pinhole diameter to 1 Airy unit for each emission channel.
Institute for Biochemistry, Germany). The monoclonal anti-giantin and the monoclonal anti-ERGIC53 antibodies were provided by H. P. Hauri (University of Basel, Switzerland). The AlexaT M488 goat antimouse and anti-rabbit IgG (H+L) antibodies were from Molecular Probes (Eugene, OR). The anti-rabbit and anti-mouse IgG Cy3 conjugated antibodies were from Sigma. The FluoroLinkT MCyT M5-labelled goat anti-rabbit and anti-mouse IgG (H+L) antibodies were from Amersham Biosciences.
Time-lapse laser-scanning confocal microscopy (LSCM)

GFP-transfected cells, grown in HEPES-buffered medium on glass-bottom Petri dishes, were imaged with a ZEISS-LSM-510 confocal microscope equipped with a thermoregulation device. Movies were produced under a 63 oil-immersion objective, at a definition of 512 512 pixels, with the pinhole diameter set at its maximum value. The GFP was excited with a 488 nm laser line and imaged through a 505550 band-pass filter. For CFP, 458-nm (excitation) and 475525-nm (band pass) emission filters were used. For YFP, 514-nm (excitation) and 530-nm (long pass) emission filters were used. The line-average mode was selected, and the number of averages was set to 1. The scan speed was the maximum, and for double imaging of the same sample the multitrack configuration was applied. For photobleaching, each round consisted of 100200 iterations (depending on the area to be bleached) at maximum-intensity laser beam of the relevant wavelength. Movie files were converted to TIFF format by Graphic Converter 4.0, edited by NIH Image 1.6.2, and again converted into QuickTime format.
Cell culture and transfection

COS7 cells were grown in DMEM supplemented with 10% (v/v) FCS, 100 U ml1 penicillin, 100 g ml1 streptomycin and 2 mM L-glutamine. RBL cells were grown in MEM supplemented with 16% (v/v) FCS, 100 U ml1 penicillin, 100 g ml1 streptomycin and 2 mM L-glutamine. NRK cells were grown in DMEM supplemented with 5% (v/v) FCS, 100 U ml1 penicillin, 100 g ml1 streptomycin, 2 mM L-glutamine and nonessential amino acids. COS7 and NRK cells were plated on glass coverslips the day before transfection. They were transfected with the Trans Fast Transfection Reagent (Promega, Madison, WI) in accordance with the manufacturers instructions. Alternatively, resuspended COS7 cells were transfected with the Gene Pulser Electroprotocol (Bio-Rad, Hercules, CA) in accordance with the manufacturers instructions. The cells were treated 16 h after transfection.
DNA constructs
Cloning of human GM130 cDNA. Poly(A)+ RNA prepared from QGP-1 (human pancreatic carcinoma) cells was used to construct a complementary DNA library in APII bacteriophage. A golgin-95 cDNA fragment46 was prepared by reverse transcription polymerase chain reaction (RT-PCR) from QGP-1 RNA to obtain a probe for screening. The positive clones were subcloned into the pBluescript SK plasmid vector. The cDNA clone with the longest insert (3.8 kb, named QSY1111) was subcloned and subjected to nucleotide sequencing. To obtain the full-length cDNA, we constructed another cDNA library using a poly(A)+ RNA fraction enriched with 4 kb RNAs that can hybridize with a fragment of QSY1111. Screening 1106 independent clones of the new cDNA library yielded 60 positive clones. The clone with the longest insert (4.3 kb) was subcloned into the pSG5 vector (Novagen, Madison, WI) and sequenced. Full-length human GM130 constructs. These constructs were generated by in-frame insertion of the EcoRIPmaCI cDNA fragment of pSG5/GM130 into EcoRI/SmaI-digested pEGFP-C2/pEYFP-C2 vector (Clontech, Palo Alto, CA) or into the EcoRI/EcoRV-digested pcDNAIII1AB vector, which encodes the Flag epitope. Assessment of the p115-binding activity of GFPGM130. Because the GFP protein was inserted at the N terminus of GM130, and this region is involved in p115 binding, we determined whether the recombinant protein was able to bind p115 with an affinity comparable to that of the endogenous GM130 protein. To this end, COS7 cell lysates from cells transfected with an empty vector and cells transfected with GFPGM130 were either immunoprecipitated with anti-p115 antibodies or passed through an affinity column on which the p115-600CGST fragment, which contains the GM130-binding site, was immobilized. In brief, cells transfected with an empty vector or with the GFPGM130 construct were treated with lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton-X 100, 1 mM EDTA and protease inhibitors). Cell lysates were incubated with preimmune or specific antibodies overnight at 4 C, then with Protein ASepharose for 1 h at 4 C. The immunoprecipitates were washed extensively (7 in lysis buffer) and analysed by SDS PAGE. Alternatively, cell lysates, prepared as described
Quantification of colocalization
Confocal images of double- or triple-labelled COS7 cells were acquired, exported as JPEG files, and analysed for fluorescent signal overlap. Using Adobe Photoshop 5, a grid (each grid square covered 1% of the image) was projected onto the image of each channel, and all fluorescent structures were counted. Signals were considered as individual structures if they consisted of spots of intensity values >50 (in a range of 0255). Then the merged images were analysed and the colocalizing structures were counted. At least 10 cells for sample were analysed, counting from 15 to 250 structures (depending on the examined marker) per cell.
Correlative light immuno-electron microscopy (CLEM) and immuno-electron microscopy

COS7 cells were grown on CELLocate coverslips (Eppendorf, Germany) in DMEM supplemented with 10% FCS and 2 mM L-glutamine. Cells were fixed with 0.05% glutaraldehyde plus 4% paraformaldehyde in 0.2 M HEPES (pH 7.4) for 15 min, and then with 4% paraformaldehyde in the same buffer for 30 min. After washing, cells were incubated in 0.2% saponin dissolved in blocking solution (PBS supplemented with 1% BSA and 50 mM NH4Cl) for 30 min, then with polyclonal anti-GM130 antibodies overnight, in blocking solution, washed and treated with the anti-rabbit IgG conjugated with CY3 (Sigma) for 60 min. After washing, samples were analysed under LSCM and the cell of interest underwent optical sectioning along the Z-axis, when its position within the coordinated grid was determined. Next, samples were incubated with nanogold-conjugated Fab fragments of anti-rabbit IgG (NanoProbes, Yaphank, NY) diluted in blocking solution (1:100) for 60 min, extensively washed and fixed with 1% glutaraldehyde in 0.2 M HEPES (pH 7.3) for 5 min. Gold particles were enhanced by GoldEnhancer (NanoProbes) in accordance with the manufacturers instructions. Routinely, the time of enhancement was about 46 min. After washing samples were treated with 1% osmium tetroxide
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plus 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.3) for 2 h on ice (all incubations before this step were performed at room temperature) in the dark, then dehydrated, embedded in Epoxy resin, and polymerized for at least 24 h. The CELLocate coverslips were then dissolved with 40% hydrofluoric acid, and the samples were intensively washed with buffer and then distilled water. Finally, the cell of interest was sectioned tangetially. Serial sections (of 100 nm) were collected on slot grids covered with formvar-carbon supporting film and examined at 80 kV in a Zeiss 109 electron microscope. The images collected by LSCM and EM were aligned with Adobe Photoshop, and the structure of interest was identified on the basis of its position in space and its labelling with colloidal gold. For the immunoEM colocalization of HP lectin and GM130 a double-labelling protocol was performed using diaminobenzidine reaction and silver enhancement. RBL cells were fixed and permeabilized as described above and incubated with the anti-GM130 polyclonal antibody. Next, cells were washed and treated with nanogold-conjugated Fab fragment of anti-rabbit IgG (NanoProbes). Gold particles were enhanced by Silver Enhancer (Aurion, Wageningen, The Netherlands) in accordance with the manufacters instructions. The enhancement reaction was developed for 60 min at 20 C in the dark. After washing, cells were incubated with HRP-conjugated HP lectin (diluted 1:20 in blocking solution) overnight. Afterwards, cells were washed, fixed in 1% glutaraldehyde in phosphate buffer (pH 7.4) for 5 min and treated with diaminobenzidine and H2O2 for 10 min at room temperature in accordance with ref. 25. After washing, samples were treated with 0.5% osmium tetroxide plus 0.75% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.3), dehydrated, and embedded in Epoxy resin. Serial sections (70 nm) were collected on copper grids and examined at 80 kV in a Zeiss 109 electron microscope. CryoimmunoEM labelling was performed in accordance with ref. 49.
RECEIVED 17 APRIL 2001; REVISED 17 JULY 2001; ACCEPTED 7 SEPTEMBER 2001; PUBLISHED 9 NOVEMBER 2001.
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ACKNOWLEDGEMENTS We thank P. Hauri, J. Gruenberg, B. L. Tang, J. Lippincott-Schwartz and F. Barr for antibodies, C. P. Berrie for comments on the manuscript, and Y. Misumi for G95 constructs. Supported in part by grants from Telethon (E.732), the Human Frontier Science Programme (to M.A.D.M.), the European Community (EU grant No. HPRN-CT-200000081), the Italian National Research Council (99.00127.PF49 and 99.00133.PF49), and the Italian Association for Cancer Research (AIRC, Milan, Italy). The human GM130 nucleotide sequence data has been submitted to the EMBL/GenBank/DDBJ data libraries with accession number D86427. Supplementary Information is available on Nature Cell Biologys website (http://cellbio.nature.com).
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Movie 1 Dynamics of GFP-GM130. Time laps images from COS7 cells transfected with GFP-GM130. The numbers in the bottom right corner designate frame numbers and time elapsed in h.mm.ss., respectively. Movie 2 Dynamics of GFP-GM130. Time laps images from COS7 cells transfected with GFP-GM130. The numbers in the bottom left corner designate frame numbers and time elapsed in h.mm.ss., respectively. Movie 3 Dynamics of YFP-GM130 and CFP-VSV-G upon release from the 15 C block. Time laps images images from COS7 cells transfected with YFP-GM130 (red) and CFP-VSV-G (green) showing the fluorescence recovery after photobleaching of the central Golgi area (white circle) upon release from the 15 C block. Movie 4 Dynamics of GFP-G95. Time laps images from COS7 cells transfected with GFP-G95. The numbers in the bottom left corner designate frame numbers and time elapsed in h.mm.ss., respectively.

The GM130 and GRASP65 Golgi Proteins

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The GM130 and GRASP65 Golgi Proteins

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NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com

2001 Macmillan Magazines Ltd

NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

2001 Macmillan Magazines Ltd

Time-lapse laser-scanning confocal microscopy (LSCM)

Cell culture and transfection

Correlative light immuno-electron microscopy (CLEM) and immuno-electron microscopy

NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com

2001 Macmillan Magazines Ltd

NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com

2001 Macmillan Magazines Ltd

NATURE CELL BIOLOGY VOL 3 DECEMBER 2001 http://cellbio.nature.com

2001 Macmillan Magazines Ltd

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