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REVIEWS Inheritance and biogenesis of organelles in the secretory pathway Martin Lowe* and Francis A. Barr‡ Abstract | In eukaryotic cells, cellular functions are compartmentalized into membranebound organelles. This has many advantages, as shown by the success of the eukaryotic lineage, but creates many problems for cells, such as the need to build and partition these organelles during cell growth and division. Diverse mechanisms for biogenesis of the endoplasmic reticulum and Golgi apparatus have evolved, ranging from de novo synthesis to the copying of a template organelle. The different mechanisms by which organelles are inherited in yeasts, protozoa and metazoans probably reflect the differences in the structure and copy number of these organelles. Eukaryotic cells have membrane-bound organelles in order to compartmentalize and organize cellular functions. This enables a diverse range of different environments to coexist within a single cell, and has enormous implications in terms of the diversity of functions that can be carried out. At the same time as proteins are being degraded in the acidic environment of the lysosomes, new proteins are being synthesized in the surrounding cytoplasm and trafficked through the organelles of the secretory pathway. It is therefore not surprising that failure of this system can lead to a number of diseases in humans. This can take numerous different forms, which include congenital disorders of glycosylation, specific mutations in proteins unable to fold correctly and achieve their normal localization, or mutations in proteins required for secretory pathway function1,2. The problem with possessing membrane-bound organelles such as the endoplasmic reticulum (ER) and Golgi apparatus is that they have to be maintained. In nondividing cells this is a simple housekeeping task — lipid and protein biosynthesis are associated with the ER, and the required components can then be delivered to the other parts of the system, such as the Golgi, by vesicular trafficking. However, what happens when cells divide? Do they create additional copies of their organelles in the daughter cells by dividing the existing copies, or build new ones de novo? Here we will discuss the different aspects of ER and Golgi inheritance, and how these involve both the division of pre-existing copies of these organelles and de novo formation of new copies. In its purest form, this means that, provided with the information encoded in the genetic material and the machinery needed to interpret this, the cell can produce the organelle with no information in the form of a template or copy of the organelle. For membrane-bound organelles, the concept of de novo synthesis needs to be modified to take into account the source of the membrane — both lipid and protein components. Therefore, a cell can create the organelles it needs to live. The alternative to de novo synthesis is the inheritance of the organelle in question, or of a template that is necessary for building the organelle. This problem can be subdivided, depending on whether the organelle is present in single or multiple copies. A single-copy organelle can be duplicated and then segregated prior to cell division, or broken down into parts that are then shared out between the two daughter cells. In theory, multi-copy organelles can simply be shared out and do not need to be dismantled. A further issue concerns the segregation or inheritance process itself. If there are sufficient copies of the organelle or fragments derived from the original intact organelle, then a stochastic partitioning mechanism can explain efficient inheritance. However, for organelles present in low copy numbers, this mechanism fails, and an active segregation process has to be invoked. Once cell division is accomplished, a growth phase then ensues. Organelles increase in amount, a process termed biogenesis, to be ready for the next cell-division event. For both single- and multiple-copy organelles, biogenesis itself can occur in two ways. Either the organelle can grow and then divide once it has reached a critical size, or it might form a template from which a new copy of the organelle is created alongside the original. It is important to note that there is nothing exclusive *Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. ‡ University of Liverpool Cancer Studies Centre, 200 London Road, Liverpool L3 9TA, UK. Correspondence to F.A.B. e-mail: doi:10.1038/nrm2179 Published online 16 May 2007 Models for organelle biogenesis and inheritance In principle, there are a number of ways in which the problem of organelle biogenesis can be solved (FIG. 1). The most simple solution is de novo synthesis. NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2007 Nature Publishing Group VOLUME 8 | JUNE 2007 | 429 REVIEWS a de novo nucleation/growth Templated assembly/growth Growth/fission Single copy Multiple copy b Cell growth Duplication Duplication Cell division Active segregation Stochastic partitioning Figure 1 | Conceptual models for organelle biogenesis and inheritance. a | In principle, there are a number of ways that organelle biogenesis can occur in proliferating cells. Biogenesis can occur by de novo synthesis, which in its purest sense means that a new copy of the organelle is generated in the absence of a template or existing copy of the organelle. Alternatively, organelle biogenesis can occur through templated assembly and growth, or through growth followed by fission. b | The principles that govern organelle inheritance will depend on how many copies of the organelle are present in the cell. A single-copy organelle can be duplicated and then segregated prior to cell division, or broken down into parts that are then shared out between the two daughter cells. Multiple-copy organelles can, in theory, simply be shared out and do not need to be dismantled. In this case, a stochastic-partitioning mechanism can explain efficient inheritance of the organelle, but for low-copy-number organelles this mechanism is not sufficient to ensure equal partitioning, and an active segregation process needs to be invoked. Centrosome The main microtubule organizing centre of animal cells. about these models. Depending on the environmental situation, life cycle or developmental stage, it might be possible to switch between de novo synthesis and inheritance of pre-existing organelles. A good example of this is provided by the centrosome, which is normally copied and then segregated between the two daughter cells3. However, in the mouse embryo, the early divisions occur in the absence of centrosomes, and these are then produced de novo prior to implantation4. how the ER is shaped in any organism, recent progress suggests that there are likely to be some common themes. Using a combination of biochemistry in a frog extract system and yeast genetics, integral membrane proteins of the reticulon family and Yop1, an integral membrane protein that interacts with the yeast reticulon Rtn1, were identified as factors able to target to and promote the formation of ER tubules7,8. Deletion of yeast Rtn1 in combination with Yop1 causes a change in cortical ER morphology from predominantly tubular-reticular to a more sheet-like cisternal morphology7,8. Reticulons are thought to adopt a hairpin structure, and it has been proposed that this promotes the formation of, or stabilizes, the tubular structure of the ER. How Yop1 contributes to ER structure is less clear. It was originally identified as an interaction partner of another integral membrane protein, Yip1, which belongs to a family of factors that are thought to be involved in the insertion of Rab GTPases into their target membranes9–11. Therefore, Rab-mediated membrane tethering and fusion events might be important for maintaining the normal organization of the ER. Several other observations regarding the organization of the ER are likely to be specific for animal cells and do not appear to apply to budding yeast. It has long been known that the extended tubular-reticular network of the ER in animal cells is aligned with microtubules12, yet is highly dynamic13,14. This is due to a combination of direct anchoring of the ER to microtubules and extension of the ER along microtubules by the kinesin-1 motor protein. Kinesin-1 can extend the ER along microtubules to form a tubular network in vitro15,16, and this is facilitated through an integral membrane protein receptor on the ER called kinectin17,18. More recent findings have identified cytoskeleton-associated protein 4 (CKAP4; also known as CLIMP63), another integral membrane protein, as a microtubule attachment factor for the ER19. This combination of extension and anchoring is probably required to maintain the ER network, as microtubules are highly dynamic structures that continuously polymerize and depolymerize. Rab GTPases Rab proteins are Ras-like GTPases that regulate membrane-trafficking events in eukaryotic cells. Different Rab proteins are specific for different transport pathways and different subcellular compartments. Kinesin-1 A member of the kinesin family of microtubule-based motors, which typically move towards microtubule plus ends. The endoplasmic reticulum In all organisms, the ER is a continuous tubular-reticular network that is connected with the nuclear envelope (NE), yet some aspects of its organization differ (FIG. 2a,b). This topic has been extensively reviewed for yeast and animal cells elsewhere5,6, and we will therefore focus on the aspects of ER structure that are most relevant to ER inheritance in yeast and animal cells here. In budding yeast, the perinuclear ER can be viewed as a subdomain of the NE, whereas the bulk of the ER, referred to as peripheral or cortical ER, lies under the plasma membrane. By contrast, in most metazoan cells, including mammalian cells, the ER forms a spread hexagonal network joined to the NE. Although little is known about Mechanism of ER inheritance in yeast Yeasts have a closed mitosis in which the NE does not break down and the mitotic spindle is inside the nucleus (FIG. 3). This has the consequence that the NE and perinuclear ER are partitioned together with the DNA through the action of astral microtubules. These position the nucleus such that it will be divided into two equal parts in the mother and daughter cells when cell division occurs20,21. In budding yeast, the fate of the cortical ER is radically different to that of the perinuclear ER. Morphological studies have revealed that the cortical pool of ER first extends into the bud and then becomes anchored at the tip of the growing bud, before expanding along the cortex to fill the bud22. The machinery for ER inheritance. Genetic approaches in budding yeast have started to elucidate the machinery required for localizing the ER to the cell cortex, and for directing its inheritance during cell division. Defects in components of the coatomer complex protein-I (COPI) pathway, required for retrograde transport from the 430 | JUNE 2007 | VOLUME 8 © 2007 Nature Publishing Group REVIEWS a c b d Figure 2 | View of the ER and Golgi in yeast and animal cells. a | The yeast endoplasmic reticulum (ER). A Saccharomyces cerevisiae cell expressing a fluorescently tagged version of the ER marker Hmg1 (left). A differential interference contrast-microscopy image (middle) and merged image (right) are shown. The nuclear ER is connected to the cortical ER network underlying the plasma membrane (large arrowhead) by cytoplasmic ER tubules (arrow). Note the presence of a tubular ER segregation structure aligned with the newly forming bud axis (small arrowhead). Scale bar represents 2 μm. b | The animal cell ER. Immunofluorescence microscopy of a Xenopus laevis cell stained for the ER marker protein disulphide isomerase (green), microtubules (red) and DNA (blue). The ER is continuous with the NE and forms an elaborate interconnected network of tubules that closely align with microtubules. Scale bars represent 10 μm (left panel) and 5 μm (right panel). c | The yeast Golgi. Pichia pastoris cells expressing a fluorescent Golgi marker Sec7 (left). A phase contrast (middle left) microscopy image and the merged image are shown (middle right). The Golgi exist as separate structures dispersed throughout the cytoplasm. Scale bar represents 2 μm. Electron microscopy reveals these to be individual stacks of Golgi cisternae (far right). Scale bar represents 200 nm. d | The mammalian Golgi. African green monkey cells (left panel) were stained for the Golgi protein giantin (green), microtubules (red) and DNA (blue). Note the single copy Golgi ribbon that is located adjacent to the nucleus near the microtubule organizing centre. Scale bar represents 10 μm. The right panels show electron microscopy images of the Golgi apparatus of human skin fibroblasts, which reveals Golgi cisternae layered on top of each other to form the Golgi stacks. Individual stacks are connected by tubules to form the Golgi ribbon. Scale bar represents 200 nm. Part b modified with permission from REF. 128 © (1999) American Society for Microbiology. Part c (left) is reproduced from Nature Cell Biology (2002) REF. 72 © (1999) Macmillan Publishers Ltd. and part c (right) is modified with permission from REF. 71 © (1999) Rockefeller University Press. Mitotic spindle A highly dynamic array of microtubules that forms during mitosis and serves to move the duplicated chromosomes apart. COPI Coat-protein complexes that are required for vesicle formation and trafficking between the endoplasmic reticulum and Golgi. Unknown function essential-1 (Ufe1). A SNARE protein involved in both fusion between endoplasmic reticulum membranes and vesicle fusion with the endoplasmic reticulum. Golgi to the ER, or the SNARE protein unknown function (Ufe1)23, alter normal ER structure from a tubular to a sheet-like morphology, with a corresponding relocation of peripheral ER away from the cell cortex24. Presumably, this is because the composition of the ER changes if recycling back from the Golgi is blocked. Consistent with this idea, mutants in the signal recognition particle that is needed to target secretory and membrane proteins to the ER, also have altered ER morphology 24. The cortical pool of tubular ER is inherited by an actin-dependent process involving the myosin V family motor Myo4 and an adaptor protein, She3 (REF. 25). Disruption of actin through the use of drugs, the deletion of Myo4 or mutations that inactivate the ATPbinding domain in Myo4 reveal that this actin- and motor-activity-dependent-pathway is required for the directed extension of ER tubules into the bud25. In addition to actin and myosin, a number of other components required for cortical ER inheritance have been identified. Ice2 (inheritance of cortical ER-2) is a multi-spanning transmembrane protein that, when essential-1 disrupted, leads to a collapse of the cortical ER in the mother cell and a failure of the cortical ER to enter the bud26. The precise function of Ice2 remains to be established, but it seems to be required for both the normal tubular morphology of the cortical ER and its transport into the bud. Mutants in the yeast auxilin Aux1 (also known as synthetic lethal with Arf1 (Swa2)) cause specific defects in the inheritance of the cortical but not the perinuclear ER or NE27. One interesting possibility to account for this finding comes from the identification of a ubiquitin-associated (UBA)-type ubiquitin-binding domain within Aux1 (REF. 28). Ubiquitin-mediated pathways have many functions at the ER, as part of the ER-associated degradation pathway that deals with misfolded proteins29, and as regulatory components of specific signalling pathways30. The role of Aux1 in cortical ER inheritance suggests that ubiquitylation might also be important for this process. This idea is supported by observations that the ubiquitin-dependent chaperone Cdc48 (cell division cycle-48; homologous to the mammalian protein p97) is required for remodelling the nucleus and ER during yeast mating31,32. NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2007 Nature Publishing Group VOLUME 8 | JUNE 2007 | 431 REVIEWS a Myo4 Cortical ER NE ER tubule Actin She3 b Nuclear envelope Mitotic spindle NE c Cortical ER Centrosome/spindle pole body ER tubule Exocyst Chromosomes Actin Exocyst A multisubunit protein complex that is important for docking secretory vesicles with the plasma membrane, and anchoring the endoplasmic reticulum to the plasma membrane during cell division. Figure 3 | Inheritance of the ER in yeast. a | In Saccharomyces cerevisiae, the nuclear envelope (NE) is connected by tubules to the cortical endoplasmic reticulum (ER) network, which is located close to the plasma membrane. b | Inheritance of the NE takes place during mitosis. Microtubules elongate inside the nucleus along the mother–bud axis to form the mitotic spindle, pushing the NE into the bud. The NE is therefore segregated alongside the chromosomes between the mother and daughter cells. c | The cortical ER uses the actin cytoskeleton for its inheritance. ER tubules move along actin cables across the mother–bud axis using the Myo4 myosin motor protein, which is attached to the ER membrane by its adaptor protein, She3. After reaching the bud tip, the ER tubule is anchored by the exocyst subunit Sec3. Another exocyst subunit, Sec6, also appears to have a role in cortical ER inheritance, perhaps by binding to the ER reticulon protein Rtn1 (not shown). Following attachment to the cortex at the bud tip, ER tubules subsequently spread along the cell periphery to form the lattice-like cortical ER network in the daughter cell. Sec61 translocon An endoplasmic reticulum (ER)-localized protein complex that facilitates the insertion of newly synthesized secretory and membrane proteins into the ER. Cdc42 A small Rho-family GTPase that regulates localized actin dynamics in cells. CDK1 The catalytic subunit of the principal serine/threonine kinase that regulates entry into mitosis. B-type cyclin The regulatory subunit of the principal mitotic serine/ threonine kinase. In mammals, cyclin B binds to the catalytic subunit CDK1. Cdc28–cyclin-2 A serine/threonine kinase that regulates entry into S phase, comprising a catalytic subunit (Cdc28) and a regulatory subunit (cyclin-2). Landmarks for ER inheritance. Once cortical ER tubules enter the bud, they become anchored at the bud tip, and this process requires the function of the exocyst, a multi subunit tethering complex required for polarized transport of secretory vesicles into the bud33,34. A number of exocyst subunits have been linked to ER inheritance, one of which is Sec335–37. Sec3 is not an essential component of the exocyst, and in its absence cells are still capable of secretion35. However polarization of the bud is altered, and inheritance of the cortical ER but not the Golgi or mitochondria is defective35. Conversely, overproduction of Sec3 appears to promote the capture of ER in the bud, and it has therefore been proposed that this is a key component of the inheritance machinery that might function as a spatial landmark for cortical ER inheritance35. Significantly, another exocyst subunit, Sec6, was identified as an interaction partner of Rtn1 (REF. 8), which as discussed above functions in shaping the cortical ER7. Therefore, Rtn1 might form part of a receptor on the ER that can interact with the exocyst and promote anchoring of the cortical ER at the bud tip. Intriguingly, the exocyst can also interact with the Sec61 translocon, and this might form a further way to link the ER to the bud tip38. Although failing to give a complete picture, taken together, these findings suggest that multiple activities associated with the ER are crucial for its normal morphology and inheritance. Regulation of yeast ER inheritance An important issue concerns the regulation and proper timing of ER inheritance during the cell cycle. After exiting mitosis, yeast cells grow throughout the G1 phase. This is reflected by the random organization of the actin cytoskeleton, and hence non-polarized delivery of secretory vesicles to the cell surface39. At the G1–S transition, bud formation is initiated, and this is coupled to the polarization of the actin cytoskeleton through a mechanism involving local activation of the Rho-family GTPase Cdc4239–41. Cdc42 also interacts with the exocyst and is required for the polarized delivery of secretory vesicles to the site of the forming bud42. Therefore, the yeast switches from so-called isotropic growth to budding — a form of polarized growth. This process is inhibited in mitosis and promoted during entry into S phase by different cyclin-dependent kinase complexes. During mitosis, the mitotic kinase Cdc28 (the budding yeast homologue of CDK1), which is associated with one of a number of B-type cyclins, prevents formation of a new bud43,44. At the onset of S phase, activation of Cdc28– cyclin-2 (Cln2) triggers the relocation of Cdc24, the 432 | JUNE 2007 | VOLUME 8 © 2007 Nature Publishing Group REVIEWS G2 ER network NE Prophase Metaphase Microtubule Centrosome G1 Chromosomes Mitotic spindle Anaphase Telophase Figure 4 | Inheritance of the ER in mammalian cells. In interphase, the endoplasmic reticulum (ER) extends throughout the cytoplasm as an interconnected network of tubules (purple), organized by the microtubule cytoskeleton (orange). The ER is continuous with the nuclear envelope (NE). During prophase, nuclear lamins become phosphorylated, releasing the anchoring between membrane and chromatin, but the NE remains intact at this point. NE breakdown occurs as cells enter prometaphase as a consequence of microtubule-induced tearing, and NE membrane components become dispersed throughout the metaphase ER network (green ER network). Connections between the ER and microtubules are lost in mitosis owing to the phosphorylation of proteins that link these structures. ER partitioning into the newly forming daughter cells is ensured by the spreading of the network throughout the cytoplasm of the dividing mother cell. During telophase, the NE begins to reform around the segregated chromosomes, ultimately becoming continuous with the extended ER network, which itself has re-attached to the microtubule cytoskeleton. Cell-biological studies have shown that microtubules and the motor protein dynein work together to peel the NE and associated ER from the surface of the chromatin53,54. This requires the action of mitotic kinases such as cyclin-dependent kinase-1 (CDK1), which results in the disassembly of the nuclear lamina and releases the anchoring between membranes and chromatin50,55. Once released from the chromatin, what happens to the ER? As discussed above, the ER is attached to and moved along microtubules, but this does not seem to be relevant for ER inheritance. Rather, the attachment between the ER and microtubules appears to be lost, in part owing to the phosphorylation of CLIMP63 in mitosis19. Consistent with this idea, the ER is excluded from the region of the cell that is occupied by the mitotic spindle and chromosomes, but spreads throughout the rest of the cell volume52. Dynein A multisubunit microtubulebased motor, typically moving towards microtubule minus ends. guanine exchange factor for Cdc42, from the nucleus to the cell cortex in the region of the presumptive bud site. Here it promotes Cdc42 activation and subsequent actin polarization45. Because ER inheritance is actin dependent, this process will start only once the actin cytoskeleton has polarized and bud formation has commenced at the G1–S transition. Recent observations have indicated that a mitogenactivated protein kinase (MAPK) pathway activated by cell-wall integrity defects can control ER inheritance in yeast46. Although it is not immediately apparent how this relates to the cell-cycle control of bud formation, there is an intriguing link between these two processes. Environmental stresses can cause perturbations in the actin cytoskeleton, and this triggers a delay in bud formation while the cell adapts to these altered conditions47. It has therefore been proposed that ER inheritance is linked to sensing of osmotic stress and cell-wall integrity sensing pathways46,48. These signalling mechanisms together with active actin–myosin-dependent transport couple the inheritance of the cortical ER with the process of bud formation in yeast. ER inheritance in mammalian cells. In mammalian cells, the NE breaks down in mitosis and NE integral membrane proteins become spread throughout the ER49,50. However, although undergoing large structural changes, the ER is not actually broken down into fragments or vesicles (FIG. 4). Measurements of long-range diffusion within the ER show that this is unaltered in mitosis. This finding suggests that the reticulum remains intact, and that what is happening is simply a topological change51,52. Nuclear lamina A structure composed of lamin intermediate-filament proteins that is important for integrity of nuclear envelope. Golgi matrix A biochemically defined meshwork of proteins that retains the characteristic cisternal shape of Golgi membranes. The Golgi apparatus The Golgi apparatus is a cisternal array of membranes, either highly organized into a stack as in plants, animals and protozoa, or a more dynamic arrangement of free cisternae and stacks as seen in fungi (FIG. 2c,d). Mammalian cells organize these stacks into a single-copy array or ribbon in interphase cells, whereas plants and flies have multiple single stacks dispersed throughout the cytoplasm. These different types of organization seem to be important in terms of function. During development in Drosophila melanogaster, it is possible for a single cell to use different groups of Golgi stacks, termed exocytic units, to transport a selected set of cargo proteins to a defined destination on the cell surface. This creates a polarized signal that is important for controlling the development of the anteroposterior and dorsoventral axes of the oocyte56. Mammalian cells, with their functionally single-copy Golgi, cannot do this and must therefore solve similar problems in a different way, perhaps by additional specific membranetrafficking pathways. Some of these differences reflect the different interactions of Golgi with the cytoskeleton in different organisms. The perinuclear Golgi ribbon seen in mammalian cells is explained by interactions with the microtubule cytoskeleton and the activities of dynein and other microtubule-dependent motors57,58. Yeast genetics and cell-biological approaches in mammalian cells have identified many factors that are required for normal Golgi organization, and this has been reviewed in detail elsewhere59–61. It is striking that many of the components identified seem to be components of the transport machinery, which suggests that continuing vesicular transport is a key factor in determining Golgi structure62. Electron microscopy and biochemical approaches indicate that there is a structural matrix (the Golgi matrix) of proteins analogous to the nuclear lamina at the surface of the Golgi cisternae, and this has been implicated in Golgi stack and ribbon formation63–68. However, it is now clear that no single component acts alone to mediate Golgi organization; rather, this matrix is made up of many redundant components acting in different pathways. Indirect support for this idea is provided by the identification of a mammalian cell line that is temperature sensitive for secretion VOLUME 8 | JUNE 2007 | 433 NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2007 Nature Publishing Group REVIEWS a 2:12 4:12 6:00 11:00 13:54 conclude that lamin A or C are not important structural components of the nucleus. Mutations or loss of lamin A or C result in subtle alterations to the properties of the nucleus, and this manifests itself in the form of a number of human diseases — termed laminopathies — that include Hutchinson–Gilford progeria, a premature ageing syndrome70. Existing Golgi elements (red) adjacent to ER exit sites (green) De novo assembly of ER exit sites, closely followed by a new Golgi b Nucleus Golgi Basal bodies c Anterior Posterior Figure 5 | Golgi biogenesis and inheritance in yeast and protozoa. a | Top panels show images captured from a movie of living Pichia pastoris yeast cells expressing fluorescently labelled markers for endoplasmic reticulum (ER) exit sites (Sec13–GFP, in green) and the Golgi apparatus (Sec7–DsRed, in red). Note the de novo appearance of two ER exit sites closely followed by new Golgi structures in close proximity to them (arrowheads). The schematic view underneath illustrates the de novo formation of ER exit sites (green) and Golgi structures (red) in the mother cell and in the newly forming bud that will ultimately form the daughter cell. Golgi inheritance occurs by de novo synthesis in the bud. Late Golgi elements are transported into the bud separately from other Golgi components (not shown). b | Protozoa such as Trypanosoma brucei typically have a single-copy Golgi that lies adjacent to the basal body (left). Trypanosoma brucei carries out de novo Golgi assembly adjacent to the existing Golgi apparatus. Quite how this is brought about is currently unclear, but recent work suggests that the centrin-2 protein may be required to guide new Golgi synthesis. c | Centrin-2 (green) is located in pools at the Golgi (indicated by red fluorescent Golgi marker, closed arrow heads) and basal body (arrow). At an early time point, a pool of centrin-2 is present next to the existing Golgi (left), and this marks the site at which the new Golgi forms (right). Centrins may therefore form a template structure that is capable of initiating de novo biogenesis of the Golgi apparatus. Part a reproduced with permission from Nature Cell Biology REF. 72 © (2002) Macmillan Publishers Ltd. ER exit site A specialized site in the endoplasmic reticulum (ER) where vesicles are generated that transport proteins from the ER to the Golgi. Basal body The microtubule organizing centre of protozoa, equivalent to the centrosome of animal cells, typically the nucleating site for flagella and cilia. Centrin A calcium-binding protein that is associated with microtubule organizing centres. and growth owing to the lack of Golgi matrix protein of 130 kDa (GM130)69. GM130 has been implicated in shaping Golgi cisternae and in the assembly of Golgi stacks into a ribbon-like array64,65,68, yet in its absence cells appear to have a functional Golgi apparatus and grow normally at 34°C (REF. 69). Exactly what this means can be debated, but it does suggest that GM130, and by extension other Golgi matrix proteins, contribute to Golgi organization and oppose the disruptive effects of thermal and perhaps other stresses. However, they might not be essential under all conditions. A parallel could be drawn here with the role of lamins in the NE. Whereas lamin B is essential for NE integrity and morphology, lamins A and C are dispensable for this both in vitro and in vivo. However, it would be wrong to Diverse mechanisms of Golgi biogenesis Reflecting the different types of Golgi organization in different organisms, there seems to be diverse mechanisms of Golgi biogenesis — from de novo synthesis to templated assembly (FIG. 1). In yeast, Golgi biogenesis is linked to the organization and function of the ER, since it forms de novo at ER exit sites71,72 (FIG. 5a). There is a mounting body of evidence to support this idea, most importantly the imaging of de novo Golgi formation by the Glick and Nakano laboratories73,74. A further example of de novo Golgi formation can be found in the protozoan Trypanosoma brucei, which has a single-copy Golgi apparatus that lies adjacent to the basal body75 (FIG. 5b). T. brucei build a new Golgi de novo adjacent to the basal body by a combination of de novo synthesis and transfer of material from the old copy75–77. Simultaneously, the single ER exit site is also duplicated, which suggests these two events are co-ordinately regulated, or reflect a single process. A caveat is that it is difficult to discriminate between true de novo assembly and templated growth that requires the original copy of the Golgi apparatus. Although little is known about the molecular details of these processes, recent work suggests an intriguing link to the centrin family of calcium-binding proteins; centrins are associated with microtubule organizing centres in many organisms, including trypanosomes. T. brucei has two centrins. The first of these is exclusively localized to the basal body and required for its duplication, whereas the other, centrin-2, is associated with a novel structure adjacent to the Golgi apparatus and is required for Golgi duplication (FIG. 5c)78. As discussed elsewhere, de novo synthesis is not the only way to build a new Golgi apparatus (FIG. 1), and other protozoa use a radically different mechanism. Toxoplasma gondii is an obligate intracellular eukaryotic parasite of medical significance that also serves as a good model system for the study of membranetrafficking processes and organelle function79. In this organism, the Golgi apparatus is a single-copy organelle that grows by a lateral extension process, and then undergoes medial fission during cell division, so that each daughter cell obtains a functional Golgi80. A similar mechanism has also been seen in other protozoa such as Trichomonas81. In mammals it is different again. Throughout S phase, when cells grow, new material is continuously delivered to the pre-existing Golgi. Recent findings indicate that this is a size control mechanism that is tightly coupled to the synthesis and delivery of new Golgi enzymes from the ER62. As in yeast, the delivery of material from the ER is therefore crucial for the establishment and maintenance of the Golgi, and it is not surprising that Golgi in animal cells also have an intimate relationship with ER exit sites. 434 | JUNE 2007 | VOLUME 8 © 2007 Nature Publishing Group REVIEWS G2 Nucleus Prophase Mitotic Golgi fragments Metaphase Golgi ribbon Golgi biogenesis G1 Telophase ER Golgi inheritance Anaphase Figure 6 | Inheritance of the Golgi in mammalian cells. The interphase Golgi ribbon is composed of stacked cisternae that are linked by lateral tubular connections to form the Golgi ribbon, which is often located in the perinuclear region of the cell. During prophase, the lateral connections are lost as the Golgi ribbon is converted into individual stacks that remain close to the nucleus. From prophase through to metaphase, stacking of Golgi cisternae is lost, and the cisternae are converted into small ~50–70 nm vesicles, and larger vesicular and tubular elements. These mitotic Golgi fragments function as the units of partitioning, and then become dispersed throughout the cytoplasm. An alternative view is that the Golgi fragments fuse with the endoplasmic reticulum (ER), which merges these two compartments and Golgi proteins are partitioned with the ER. In telophase, the Golgi fragments fuse with each other to initiate the reformation of new Golgi stacks that ultimately connect to form a Golgi ribbon in each daughter cell. the content of the vesicle, as structural components of the Golgi do not seem to recycle to the ER, whereas Golgi enzymes and cargo can to some extent94–96. So, despite the controversy, is the mechanism of inheritance in mammalian cells so different from the other organisms discussed? Ribbon splitting is actually analogous to the binary fission mechanism used by protozoa, and might therefore involve the same underlying machinery. Rebuilding a Golgi during mitotic exit has many similarities with de novo Golgi biogenesis in yeast. It is clearly important to re-establish transport from the ER to form a new Golgi, which suggests that the delivery of ER-derived material is necessary. However, the large pool of vesicles in the cytoplasm derived from the old Golgi needs to assemble together with this to build new functional Golgi in the two daughter cells. The question is do these vesicles reflect a template for Golgi assembly, or are they simply a supply of components directed by the ER-derived material to incorporate into a new Golgi? In both cases, Golgi proteins must have self-assembly properties and form structures specifying cisternal shape and size, and stack organization. The difference is whether or not a pre-existing Golgi is needed to template Golgi formation as in protozoa, or if it can occur de novo as in yeast. Strategies for Golgi inheritance The mechanisms of Golgi inheritance reflect the different modes of biogenesis discussed above. As yeast produce a functional Golgi de novo it is not strictly essential to inherit old Golgi. However, this highlights the importance of ER inheritance, as without a properly functional complement of ER the cell will be compromised in its ability to produce Golgi de novo82. Budding yeast are also capable of targeting Golgi elements to the bud using an actin–myosin-dependent partitioning mechanism83, so in reality a combination of inheritance and de novo synthesis might be the most efficient option. Some protozoa split their duplicated Golgi down the middle, by a binary fission mechanism that reflects their mode of cell division80,81. In mammalian cells, it is clear that the Golgi breaks down into many small vesicles (FIG. 6). However, there are two possibilities to explain what happens next, and the debate continues84. The initial events as cells enter mitosis are not disputed. This includes the release of many peripheral membrane proteins into the cytoplasm and breakdown of the ribbon structure, followed by the unstacking and fragmentation of the individual cisternae into small vesicles. These Golgi vesicles might then either remain as dispersed independent entities throughout mitosis that partition according to a stochastic mechanism52,85–89, or deliver their content back to the ER90–92. Once delivered back to the ER, Golgi proteins would be trapped and partition together with the ER, as ER to Golgi trafficking is blocked at an early stage for the duration of mitosis93,94. There is evidence for both ideas, and it might depend on NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2007 Nature Publishing Group Regulation of Golgi inheritance The transitions between the different phases of the cell cycle are controlled by protein phosphorylation and ubiquitylation. It is therefore not surprising that the inheritance of the Golgi apparatus is controlled by these same mechanisms. Although protein phosphorylation has historically been the focus of attention, more recent evidence points to the importance of reversible ubiquitylation in controlling the assembly state of the Golgi. Phosphorylation. Most information about phosphorylation is known for mammalian cells, in which it has been shown that, similar to the NE, CDK1 drives the breakdown of the Golgi apparatus97–99. However, CDK1 does not function alone and a number of other protein kinases of the polo and MAPK family have also been implicated (TABLE I). These kinases phosphorylate multiple proteins that are required for trafficking and Golgi structure (TABLE I), and as a consequence the Golgi then disassembles. This disassembly is in part mediated by the action of the COPI vesicle formation pathway, which consumes Golgi cisternae into vesicles97, 98. As mentioned above, ER to Golgi trafficking is blocked in mitosis, which is likely to have an important role in the reorganization of the Golgi, as under these conditions the Golgi will no longer be supplied with new components. How this transport block is achieved is still not completely understood. Multiple components of the ER to Golgi transport machinery, including the GTPase RAB1 and GM130, become phosphorylated in mitosis66,99–101, but no single crucial component has been identified. GM130 phosphorylation alone is unlikely to be the critical event, as when it is depleted from interphase cells the efficiency of ER to Golgi transport is only slightly reduced102. Rather than a single key substrate, this supports a model in which phosphorylation of multiple components reduces the efficiency of ER to Golgi transport. Other aspects of Golgi fragmentation, such as the breakdown of the Golgi ribbon, VOLUME 8 | JUNE 2007 | 435 REVIEWS Table 1 | Kinases and substrates implicated in Golgi inheritance in mammals Kinases CDK1 Interaction partners Cyclin B Substrates GM130 GRASP65 RAB1 p47 NIR2 Proposed function of substrate Golgi membrane and vesicle tethering Membrane tethering and cisternal stacking Golgi membrane and vesicle tethering Membrane fusion Phospholipid transfer Membrane tethering and cisternal stacking Golgi membrane and vesicle tethering Not applicable Refs 99,101 105,129–131 100 117 126 105,109,129–131 105 132,133 PLK1 PLK3 GRASP65 RAB1 Giantin MEK1 ERK2 PLK3 PLK3 Unknown Unknown GRASP65 RAB1 Unknown MEK1 ERK1/2 ERK1c Unknown Unknown GRASP55 Unknown Golgin-84 Not applicable Cisternal stacking Not applicable Golgi-membrane and vesicle tethering 133–136 133,137 138 103 CDK1, cyclin-dependent kinase-1; ERK1/1c/2, extracellular regulated kinase-1/1c/2; GM130, Golgi matrix protein of 130 kDa; GRASP55, Golgi reassembly and stacking protein of 55 kDa; GRASP65, Golgi reassembly and stacking protein of 65kDa; MAP kinase, mitogen-activated protein kinase; MEK1, MAP kinase/ERK kinase kinase-1; NIR2, PYK2 amino-terminal domain interacting receptor-2; PLK1/3, polo-like kinase-1/3. Golgin-84 A member of a diverse family of coiled-coil proteins that have been implicated in vesicle trafficking and shaping the Golgi. GRASP (Golgi reassembly stacking protein). A family of proteins identified in an in vitro biochemical screen for Golgi stack formation. Polo-like kinase 1 A serine or threonine kinase that is required for centrosome and spindle function in mitosis and cytokinesis. Anaphase promoting complex/cyclosome A ubiquitin ligase that is required for progression through mitosis. might require additional factors. For example, what splits mammalian-cell Golgi ribbons into single stacks or a protozoan Golgi down the middle, and is there an activity to promote this? Alternatively, this stack splitting might be a passive process explained by phosphorylation of proteins such as GM130 and golgin-84, which have been implicated in maintaining the ribbon of Golgi stacks in interphase cells68,103. Similarly, loss of the cisternal architecture of the Golgi is thought to involve phosphorylation of components linking Golgi cisternae together, and factors such as Golgi reassembly stacking proteins (GRASPs), which are targets for CDK1, have been proposed to contribute to this process66,104,105. Interestingly, the processes of Golgi ribbon and stack formation might be linked, as GRASP65 is an interaction partner of GM130 (REF. 106). On the basis of a number of observations, it has been suggested that Golgi-stack formation is a non-productive form of vesicle tethering that does not lead to membrane fusion, but rather holds Golgi cisternae together66,107. The roles of polo-like kinase-1 (PLK1) and MEK1 (MAPK and ERK kinase-1) in Golgi fragmentation are unclear, and they do not appear to be essential for Golgi breakdown. However, both have been implicated together with GRASP55 and GRASP65 in an organelle-breakdown checkpoint105,108–110. Just as unattached chromosomes signal to prevent cell-cycle progression, so too might the Golgi through GRASPs, MEK1 and PLK1105,108–111. Some evidence also exists for this in yeast, in which Grh1 (the yeast homologue of GRASP65) was found in a screen for mitotic checkpoint deficiency112. However, this is controversial, and other evidence shows that Grh1 is a Golgi protein that, as in mammals, has a function in ER to Golgi transport113. Intriguingly, recent evidence indicates there might be a link between the GRASP checkpoint and centrin-2 function in mitosis in animal cells111, which suggests that centrins may have a general function in Golgi inheritance. Ubiquitylation. In addition to phosphorylation, other types of regulatory modification are important for controlling the passage through mitosis, and perhaps the most important of these is ubiquitylation114. In recent years, a number of lines of evidence have pointed to the role of ubiquitylation in remodelling the ER and Golgi during mitosis. This has mainly emerged from studies of the ubiquitin-dependent chaperone p97 (also known as valosin-containing protein (VCP)) and its interacting proteins p37, p47 and VCIP135 (VCP/p47 complex interacting protein-1)115–121. Interestingly, p47 is a CDK1 substrate, and this regulation appears to couple these two systems for controlling the Golgi, as mutant forms of p47 that can no longer be phosphorylated by CDK1 prevent complete fragmentation of the Golgi during mitosis117. Although a major function of ubiquitylation is to target proteins for degradation by the proteasome, it is now appreciated that it also has regulatory functions, and evidence is emerging that this regulatory role is important for Golgi inheritance. The most direct evidence for this comes from a study showing that the p97–p47 cofactor VCIP135 is a deubiquitylating enzyme that is required for reformation of the Golgi complex in an in vitro assay. This suggests that ubiquitylation — through cycles of adding and removing ubiquitin to substrates — is necessary for Golgi reassembly, rather than ubiquitin-dependent proteolysis 120. A number of outstanding questions remain about the role of ubiquitylation: the first concerns the identity of the ubiquitin ligase that is responsible for carrying out ubiquitin conjugation in organelle inheritance, and the second concerns the identity of the proteins targeted by this system. An obvious answer to the first question would be the anaphase promoting complex/cyclosome, as this is a key mitotic regulator that is capable of modifying specific sets of substrates required for entry into and exit from mitosis114,122. 436 | JUNE 2007 | VOLUME 8 © 2007 Nature Publishing Group REVIEWS Cytokinesis The process of cytoplasmic division in animal cells. Answers to both these questions will be important for determining how direct the role of ubiquitin modification is, and how exactly Golgi inheritance is regulated by ubiquitylation. Outlook and future directions Despite recent advances, many aspects of the mechanisms needed to build an organelle remain mysterious. How and why do Golgi membranes adopt their characteristic cisternal shape whereas most other cellular membranes adopt vesicular or tubular morphologies? The recent identification of factors promoting ER tubulation shows this is surely protein mediated, but we still do not understand what these factors are or why this morphology is important. A second issue concerns the difference between selfassembly and de novo assembly. In general, membrane organelles self-assemble to form unique structures, that is, the protein and lipid components alone define the final form of the organelle123. Some yeasts provide an example of de novo Golgi formation, in which components selfassemble without a template to form Golgi cisternae. However, in mammalian cells and some protozoa, although these components might also self-assemble, it is unclear whether they can do so without the assistance of a template structure. This might reflect the different scale of the Golgi in yeast and mammals. In mammals, some form of templating might be needed to promote assembly, which would otherwise be too slow or inefficient during the 15–30 minute time window that is available as mammalian cells exit mitosis. Questions remain over the fate of Golgi proteins in mitosis, and this will require further investigation of multiple classes of Golgi proteins to build up a full picture of what happens to them. A related question is why does secretion stop in some organisms during mitosis but not in others? It is obvious that unicellular organisms such as yeasts might have a selective advantage if they can continue to secrete and grow throughout the cell cycle; however, it is not clear why mammalian cells stop secreting during cell division. In recent years it has become apparent that organelle function during mitosis is also important for the celldivision process itself. Asymmetrical cell divisions are required for determining cell fates during development, and it is now known that this is underpinned in part by asymmetry in the behaviour of organelles required for cell function (reviewed in REF. 124). It has been found recently that asymmetry in the activity of RAB11 associated with recycling endosomes reflects altered trafficking of molecules that specify asymmetrical cell fate 125. However, nothing is known about other organelles in this context, and there might also be differences in molecules that are associated with the ER and Golgi. Signalling molecules involved in determining cell fate have to be synthesized and delivered to the cell surface, and one might expect that this is also a point of regulation involving the ER and Golgi. Another potential mechanism of regulation is the release from organelles of components that function in the division process itself. 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ERK1c regulates Golgi fragmentation during mitosis. J. Cell Biol. 172, 885–897 (2006). Acknowledgements We would like to thank G. Warren and C. He for providing the images of dividing Toxoplasma gondii, V. Allan for images of the ER in animal cells, B. Glick for images of the Golgi in Pichia pastoris and Y. Du and S.Ferro-Novick for images of the budding yeast ER. Competing interests statement The authors declare no competing financial interests. DATABASES The following terms in this article are linked online to: Entrez Genome Project: entrez/query.fcgi?db=genomeprj Toxoplasma gondii | Trypanosoma brucei UniProtKB: Aux1 | Cdc28 | Cdc48 | CKAP4 | Ice2 | PLK1 | Ufe1 | Yip1 | Yop1 Access to this links box is available online. NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2007 Nature Publishing Group VOLUME 8 | JUNE 2007 | 439