Intracellular Trafficking – 2

BIOC 212
Winter 2019
Jason C. Young
 Compare and Contrast
• What uses GTP
• What uses ATP
• Targeting and modification signals/motifs

initiation,
signals on
membrane pinching off uncoating
cargo
attachment

COP-II

COP-I

CCV
 Stress Responses
IRE1 PERK ATF6 Heat Shock
Response
Chaperone

Activation

Signaling

Transcription
Factor and
Output
Down-
Regulation
Traffic
 Transport Through Golgi

• Golgi is organized into a stack of membranes: cis, medial, trans

• Old model: vesicles transport cargo between layers
• New model: each layer matures and becomes the next layer
– Golgi-resident proteins are carried backwards by COP-I vesicles
– clathrin-coated vesicles carry cargo to PM and endosomes
• A cytosolic protein matrix maintains organization of the stack
 Glycosylation in Golgi

• N-linked glycans are modified by removal of mannoses and addition

of different sugars, often with negative charge
• Other complex oligosaccharides are attached to Ser and Thr side
chains – O-linked glycosylation
• Many final combinations of oligosaccharides – heterogeneity
• Glycosylation protects from proteases, stabilizes protein structure

N-linked glycosylation
 Proprotein Convertases
• Some PM and extracellular proteins are made as a longer, inactive
form at the ER, then cut by proprotein convertases into a shorter,
active form at the Golgi
• Proteases recognize specific patterns of amino acids
• Cleavage often activates proteins by removing inhibitory region
• Example: proinsulin is made as one inactive polypeptide
– convertases remove middle section, the two remaining sections
form active insulin
– prevents premature signaling by insulin at the ER

Inhibitory section
Regulation by Proteases
• ATF6 and SREBP are activated by convertase
proteolysis in the Golgi
• Regulation is by trafficking
– INSIG covers ER exit signal on
SREBP/SCAP
– proteases are only in Golgi

HMG-CoAR
 Vesicle Traffic

• Multiple donor and acceptor (target) membranes in secretory

pathway
• Two mechanisms ensure that vesicles transport their contents to the
correct acceptor membrane:
– Rab GTPase proteins provide specificity of vesicle targeting and
attachment to acceptor membrane
– SNARE fusion proteins provide specificity during fusion of
vesicles with acceptor membrane
 Rab Proteins

• Large subfamily of Ras-related proteins

• Different organelles and vesicle types in secretory pathway have
unique sets of Rab proteins
• Switched “on” by GEF, “off” by GAP
• Rab-GTP binds to a large number
of Rab-effector proteins which
mediate vesicle targeting
 Rab Functions

• Rabs can act at several steps in vesicle targeting

• Assist cargo selection and coat formation during vesicle budding
• Connect vesicle to motors on cytoskeleton for transport
• Tether vesicles to acceptor membrane – specificity
• Recruit SNARE fusion proteins

Rab
Rab Rab
motor
Rab
cytoskeleton Rab
tethers

SNARE
Specific Rabs act at each vesicle transport stage
of Secretory Pathway
Golgi and early to
late endosome
Rab1: ER to Golgi Rab7, Rab9
Rab2: Golgi to ER
endocytosis from
PM to endosome
Rab5

recycling from
endosome to PM
Rab4, Rab11

exocytosis of secretory vesicles

Rab3
 Rab Membrane Anchor

• Rabs have two prenyl lipid groups attached at their

C-termini
• In the GDP-bound state, Rab is soluble and not
associated with the membrane
– lipid is covered up by other proteins (GDI, GDF)
• In the GTP-bound state, the lipid modifications are
exposed and anchor Rab to the membrane
– the Rab-effector proteins become attached to the
membrane through Rab-GTP
 Rab Activation
• Specific GEF on membrane produces anchored, active Rab-GTP
– GEF is linked to formation of vesicle coat
• Rab-GTP works through effector proteins:
– attach vesicle to motor proteins
– tether vesicle to target membrane
– activate PI kinases and GEFs to make more Rab-GTP in
clusters on acceptor membrane
soluble

prenyl lipid

anchored

Stenmark (2009) Nat Rev Mol Cell Biol 10, 513-525

 Vesicle Rab Cycle

4. 1. Vesicle Rab is activated by

GEF on donor membrane
1.
and packaged onto vesicles
– some interact with cargo,
3.
assist uncoating, or attach
to motors
2. Vesicle Rab-GTP binds
specifically to tethers on
acceptor membrane
3. After fusion,GAPs on target
membrane inactivate Rab
2. 4. Inactive vesicle Rab-GDP is
recycled through cytosol to
donor membrane
 Cytoskeleton

• Protein filaments (actin) and microtubules (tubulin) run through

cytosol and provide structure to cells
• Actin filaments: shorter, often clustered at PM, highly crosslinked
into network
• Microtubules: longer, thicker, organized around centrosome near
nucleus
• Connected to each other, and anchored to organelles and PM
(cortical cytoskeleton)

actin filaments

microtubules
 Motor Protein Transport

• Motor proteins transport vesicles along cytoskeleton

– myosins on actin, dyneins and kinesins on microtubules
– different families and mechanisms, but all are ATP-dependent
• Motors themselves do not provide targeting specificity, but bring
vesicle to general location of acceptor membrane

dyneins are AAA proteins

dynein other motors are not

Rab
 Vesicle Tethers

• Many Rab effectors are tethers – long proteins which

connect the vesicle with its acceptor
• Tethers are bound to a specific vesicle Rab on one
end, and a specific membrane site on the other end
– the site on acceptor membrane is a Rab in some
cases, different proteins in other cases
• Tethers are the first determinant of vesicle targeting
specificity

Rabs on vesicle and

target membranes
 Types of Tethers

• Tethers of different structural families act at different organelles

• Coiled-coil tethers mostly within the Golgi and on endosomes
• Multisubunit tethers act at other compartments
– ER to/from Golgi
– Golgi to PM
– Endosome and lysosome
 Coiled-coil Tethers
• Dimers with coiled-coil structures
(pairs of α-helices) that remain
assembled after use
• Coiled-coil tethers form the Golgi
matrix:
– maintain the organization of the
Golgi stack
– connect vesicles to Golgi
– anchored to membrane, or
attached by GTPases
– long filaments with multiple
Rab binding sites

Yu & Hughson Annu. Rev. Cell Dev. Biol. 2010. 26:137–56

 Multisubunit Tethers

• Most secretory pathway steps

involve multisubunit tethers
• Different structural families:
– ER to Golgi, within Golgi
(TRAPPI, II)
– endosome (HOPS/CORVET)
– ER and PM: CATCHR family
(exocyst, GARP, COG, Dsl1)
 ER to Golgi (TRAPPI)
• TRAPPI works together with coiled-coil tether p115
1. TRAPPI acts as GEF for Rab1 on Golgi
2. Rab1 binds coiled-coil tether
3. CC tether binds to vesicle and hands vesicle to TRAPPI, closer to
membrane
4. TRAPPI helps organize SNAREs

vesicle GEF SNAREs

Kim et al. 2006 Cell 127, 817–830

Lorente-Rodriguez & Barlow Cold Spring Harb Perspect Biol 2011;3:a005207
 Exocyst at PM (CATCHR Family)

1. Before tethering but after uncoating, some subunits are on either

vesicle or PM
2. Complete 8-mer complex forms tether
3. May bring SNARE to membrane to start fusion

Rab

Rab-related
GTPase
 Endosome Tethers
• CORVET: PM to endosomes, Rab5
• HOPS: endosomes to lysosomes, Rab7
• Complexes have the same core subunits
• Different end subunits bind different Rabs
• Also bind SNARES

lysosome endosome

Balderhaar & Ungermann, 2013, J Cell Sci 126, 1307–1316

 Clustering of Tethers
• Rab5 effectors have GEF or PI kinase activity on early endosome
• GEF activity produces more Rab5-GTP in local area of membrane
• PI-phosphates provide additional binding sites for vesicle tethers
• Cluster of tethers form “landing site” for vesicle
– a type of microdomain that does not involve membrane
thickness or lipid content

effector
effector

PI(3)P at endosomes
PI(4)P at Golgi
PI(4,5)P2 at PM
 Endocytosis Pathway

• Vesicle traffic between PM, early

endosome and trans-Golgi
endocytosis
• Early endosome matures into
Rab5 multivesicular body (MVB) and late
endosome
– Membrane switches from Rab5
to Rab7
• Late endosome matures into
Rab7 lysosome
• Other vesicles traffic to lysosome
 Rab Cascades
• Rab5 at endosomes activates Rab7
• Rab5 effector and tether (CORVET) is GEF for Rab7
• Rab7 effector is GAP to inactivate Rab5
• As Rab5 vesicles fuse with early endosome, more and more Rab7 is
activated, less and less Rab5 stays active
• Membrane becomes late endosome

Rab5 Rab7
 Compare and Contrast

• TRAPPI, Exocyst, endosome tethers

Rabs Sar1 (COP-II) Arf1 (COP-I)

where is the
GEF?
what are the
effectors?

when/where does
the GAP act?
before vesicle
fusion?
or after vescle
fusion?
 End of 2

• Alberts et al., Molecular Biology of the Cell

– Ch. 13, Golgi, Rab