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Blueprint Series

Complete Solution
Chapter 14

14
VESICULAR TRAFFIC, SECRETION,
AND ENDOCYTOSIS

REVIEW THE CONCEPTS
1
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Most
protein synthesis in these cells is devoted to secretion
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The cells are also
decidedly polarized with a definite gradient in organelle distribution
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After pulse-chase
labeling and sectioning the cells for electron microscopy, the sections were covered
with photographic emulsion and exposed
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In a more contemporary method, cells can be transfected with a hybrid gene encoding two proteins
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Cells expressing this gene rapidly synthesize VSVG-GFP in the ER,
which is then transported through the secretory pathway to the cell surface
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Thus, both methods require that
proteins be labeled in an early compartment so that their processing and transport can
be followed over time
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In each case, a form of microscopy was used that complements the labeling reaction
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Coat proteins play two roles in vesicle budding: 1) They provide a scaffold that
establishes membrane curvature; and 2) they interact with cargo proteins or
cargo protein receptors to provide enrichment of certain proteins in the bud
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It is the GTP-bound form of Sar or ARF that is active
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The mechanism is particularly unclear
in the case of ARF, which recruits clathrin and different adapter proteins at
different sites within the cell
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Moreover, newly formed vesicles are programmed for subsequent fusion
events by the selective inclusion of v-SNAREs and Rabs in their membrane
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In all known cases, these are
clathrin-coated vesicles
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The observation that decoating Golgi membranes by treatment of cells with
the drug brefeldin A (BFA) results in the redistribution of Golgi proteins to the
ER suggests that COPI, the major Golgi-associated coat, has a role in stabilizing
Golgi structure
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ARF1 is the small GTPase that recruits COPI to
Golgi membranes
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Since the COPI association with membranes is dynamic, this mutation would
shortly lead to uncoating of Golgi membranes
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Since GDP-restricted ARF1 produces
the same phenotype as BFA, this suggests that the drug evokes a normal physiological possibility
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Coat proteins must assemble onto a membrane to form a vesicle and must disassemble to allow the vesicle to fuse with a target membrane
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This inhibits cisternal
maturation and anterograde transport of newly synthesized proteins from the
ER to the plasma membrane
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Vesicle fusion involves two stages: first, a docking stage mediated by long coiledcoil proteins such as EEA1, and then a specific membrane fusion step mediated
by SNAREs
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Rab5 plays a role in prompting vesicle
fusion with early endosomes
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6
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Such complexes are essential in specific membrane fusion at

several stages of the secretory and endocytic pathways
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In the absence of NSF activity, vesicles bud from the ER but
are unable to fuse with downstream membranes because of the lack of vSNAREs
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7
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The procollagen bundles are
too large to fit into the vesicles that go between Golgi compartments and have
never been found in those vesicles
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8
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KDEL is a sequence feature of soluble ER luminal proteins;
KKXX is found on the cytosolic domain of ER membrane proteins
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COPI is found on the cytosolic face of the cis-Golgi membrane
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The two interact through
a bridging membrane protein, the KDEL receptor
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In the cisternal progression model, trans-Golgi proteins, for example, must be retrieved to the
medial-Golgi to generate a new trans-Golgi cisterna
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There must be interactions between COPI and Golgi proteins to promote
such retrieval
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There are four known clathrin adapter protein complexes: AP1 (trans-Golgi
to endosome), GCA (trans-Golgi to endosome), AP2 (plasma membrane to endosomes), and AP3 (Golgi to lysosome, melanosome, or platelet vesicles)
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The clathrin coat,
unlike the COPI or COPII coat, is a double-layered coat with a core coat of
adapter proteins and an external clathrin coat
...
Presently, it is not known if the coat of AP3 vesicles contains
clathrin
...

10
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Multiple
enzymes are lacking in the lysosome and the organelle becomes stuffed with
nondegraded material and therefore generates a so-called inclusion body
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This enzyme recognizes soluble lysosomal enzymes as a class and hence a defect in this protein
affects the targeting of a large number of proteins
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Similarly, defects in mannose 6-phosphate receptors would affect the targeting
of lysosomal enzymes as a class
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The trans-Golgi network (TGN) is the site of multiple sorting processes as proteins exit the Golgi complex
...
Binding is pH dependent
and occurs at the TGN pH of 6
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0–
5
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Hence, lysosomal enzymes reversibly associate with M6P receptors
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Vesicles budding from late endosomes recycle the M6P receptors back to the TGN
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This sorting is thought to be due to selective aggregation followed by budding
...
This is the case in MDCK cells, a
line of cultured epithelial cells, where there is direct basolateral-apical sorting at
the TGN cells
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Here, newly made apical and basolateral proteins are first transported in vesicles from the TGN to the basolateral surface
and incorporated into the plasma membrane by exocytosis
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The endocytosed basolateral proteins are recycled back to
the basolateral membrane
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12
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Likewise, in the TGN, newly synthesized influenza HA coat protein
is sorted into vesicles that fuse with the apical membrane and VSV G protein is
sorted into different vesicles that fuse with the basolateral membrane
...

Inhibition by a mimetic peptide but not by the mutant peptide indicates that the
VSV G protein cytoplasmic domain contains a basolateral membrane targeting
signal that most likely interacts with a component of the basolateral sorting and
targeting mechanism, and that the tyrosine is part of that signal
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Within the endocytic pathway, there is a progressive acidification (increased
hydrogen ion concentration) in compartments going from early to late endosomes to lysosomes
...
5
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At the cell surface, neutral pH, LDL
binds to LDL receptor
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5, LDL dissociates from its receptor
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Mannose 6-phosphate bearing lysosomal enzymes
dissociates from mannose 6-phosphate receptors in the acidic pH late endosomal
compartment
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Receptors become saturated with
lysosomal enzymes and the cell no longer has the capacity to direct newly

synthesized lysosomal enzymes to lysosomes
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14
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Important
mechanistic features are shared
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In multivesicular endosome for- mation, cargo proteins to be included
in the budding endosome and the Hrs pro-tein are ubiquitinated
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In closing off the
budding endosome or the budding HIV a cellular ESCRTprotein complex
recognizes the ubiquitin, and cellular Vps4 is used later to disso-ciate the
ESCRT complex
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ESCRT binds to the
C-terminal portion of HIV Gag protein
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Such a peptide might
well compete or interfere with normal cellular proteins such as ESCRT
binding to ubiquitinated Hrs
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Phagocytosis is the actin-mediated process used by some cells to engulf
whole bacteria and other large particules
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Autophagy is the process whereby a double membrane organelle or
autophagosome envelopes soluble cytosolic proteins, peroxisomes, or
mitochondria and delivers them to the lumenof the lysosome for
degradation
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16
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Once LDL has been internalized by receptor-mediated
endocytosis, the low pH of the late endosome dissociates the LDL ligand
from the LDLR, and the empty LDLR is recycled back to the plasma
membrane while the LDL is digested in the lysosome
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When the
TR is recycled back to the plasma membrane, the neutral pH of theexternal
environment dissociates the apotransferrin ligand from the TR
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The mutant LDLRs bind LDL normally but only randomly get internalized
into clathrin-coated vesicles because the mutated cytoplasmic domains fail
to interactwith the AP2 complex
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