Vesiclemediated protein transport in the secretory and endocytic pathways

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Vesicle-mediated protein transport in the
secretory and endocytic pathways
ARF
ARF
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Exocytosis (post-Golgi sorting)

• Pathways
• Lysosomal targeting
• Secretion (constitutive and regulated)
• PM protein delivery (polarized and non-polarized
  cells)
• Retention of Golgi-resident proteins
• Machinery
• Examples from the literature

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Pathways
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1. Lysosomal sorting

• Lysosomal hydrolases are modified by
  mannose-6-phospate (M6P) in the cis-Golgi
• The M6P receptor recycles between the trans-Golgi
  network and late endosomes in clathrin-coated
  vesicles (AP-1, GGA)
• The phosphate is removed from hydrolases in late
  endosomes to prevent recycling of the hydrolases
  with the M6P receptor
• Some hydrolases are secreted and are captured and
  delivered to lysosomes by endocytosis via
  PM-localized M6P receptors
• Lysosomal membrane proteins are sorted by a
  different mechanism via a sorting signal in their
  cytoplasmic tails

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2. Constitutive and regulated secretion
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Regulated secretion via secretory granules

• Occurs in endocrine, exocrine or neuronal cells
• insulin secretion in pancreatic B-islet cells in
  response to elevated blood glucose
• trypsinogen secretion in pancreatic acinar cells
• Sorting occurs by co-aggregation of proteins
• Most secretory proteins undergo proteolytic
  processing from a proprotein to the mature form
• Proteins become highly concentrated (condensed)
  -gt dense-core granules
• Exocytosis occurs in response to a trigger ex.
  Ca2

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5-48
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Electron micrographs revealing aggregation and
cleavage of proinsulin
17-41
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Regulated secretion II release of
neurotransmitters and the recycling of synaptic
vesicles (which form from endosomes)
Lodish et al. Figure 21-29
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3. Plasma membrane protein trafficking

• Delivered via membrane vesicles from the TGN to
  the cell surface
• Share same vesicles as constitutively secreted
  proteins
• More than one pathway in polarized cells (also
  probably in non-polarized cells)
• Apical lipid rafts/glycosylation/unknown
• Model proteins include GPI-anchored proteins, HA
• Basolateral cytoplasmic sorting signal/ m1b
• Model proteins include VSVG, LDLR
• Polarized sorting occurs via trancytosis occurs
  in some polarized cell types

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Baso-lateral
Apical
Lodish et al. Figure 17-43
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Components of the membrane trafficking machinery
involved in polarized transport in epithelial
cells
Mostov et al. 1999 Cell 99121-122
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4. Retention of Golgi-resident proteins

• Golgi morphology and composition is maintained
  despite the flux of proteins and lipids in the
  secretory pathway
• Requires recycling of proteins and lipids
• Also requires retention of Golgi-resident
  proteins

a. Segregation by bilayer thickness
b. Segregation by oligomerization
Golgi-resident protein
PM protein
Phospholipid- rich
Sterol/cholesterol- rich, destined for PM
After Munro (1998) Trends Cell Biol. 811-15
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Possible mechanisms for retention

• Lack of positive sorting signals for
  incorporation into post-Golgi vesicles
• Formation of large protein aggregates or protein
  immobilization (but see Cole et al 1996 Science
  273797)
• Putative retention signal in the transmembrane
  domain of some proteins
• TM domains of Golgi-resident proteins tend to be
  shorter than those of PM proteins
• If replace transmembrane domain of plasma
  membrane protein with TM domain of Golgi protein,
  is retained in the Golgi complex
• Thick TM domains are targeted to PM destined
  vesicles, thin excluded because of differences
  in lipid composition (PM has thicker membrane)

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Machinery
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Sorting and trafficking machinery in exocytosis-
a partial list

• Clathrin and accessory proteins- mediate vesicle
  formation
• Adaptors- mediate capture of different types of
  cargo
• Rabs- act as timers for vesicle targeting and
  fusion
• SNARES- vesicle targeting and fusion machinery
• Dynamin- putative pinchase
• Microtubules and motors- direct vesicle movement
• Exocyst- site of docking of exocytic vesicles at
  the plasma membrane in yeast (also mammalian
  homologs)
• Lipid rafts- putative lipid-based platform for
  apical sorting of proteins in polarized cells,
  also thought to function in endocytosis

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Clathrin

• Provides structural force to drive vesicle
  formation
• Forms 50-100 nm diameter vesicles
• Clathrin triskeleons consist of a heavy chain and
  light chain
• Bind adaptor proteins
• Coated vesicles lose their coats just after their
  formation
• Clathrin coated vesicles are not all alike-
  participate in different trafficking steps
• Endocytosis
• TGN to endosome

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Components that participate in budding of coated
vesicles
Lodish et al. Figure 17-51
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Key steps in the formation of clathrin-coated
vesicles
Kirchhausen 2000 Nature Reviews Molecular Cell
Biology 1187
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Rab proteins

• Small GTPases
• Distributed to distinct intracellular
  compartments
• Regulate transport between organelles
• Membrane tethering
• Vesicle budding
• Vesicle motility

Zerial and McBride (2001) Nature Reviews
Molecular Cell Biology 2107
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Synaptic vesicle and plasma membrane proteins
important for vesicle docking and fusion
Lodish et al. Figure 21-31
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Adaptins

• Adaptins are subunits of adaptor protein (AP)
  complexes
• 4 Types of adaptins with distinct intracellular
  localizations
• Responsible for linking cargo binding with
  clathrin recruitment
• Recognize signals in cytoplasmic tails of cargo
  proteins ex. dileucine, YXXØ (where Ø is bulky
  hydrophobic residue)
• GGA and stonins are related proteins

AdaptinsThe Final Recount Boehm and Bonifacino,
MBC 12 2907-2920
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Dynamin

• Putative vesicle pinchase
• Cytosolic protein that polymerizes at the neck of
  clathrin coated pits (and caveolae)
• GTPase
• Dominant negative mutants that cannot bind GTP
  inhibit pinching off
• Discovered by analysis of a temperature sensitive
  Drosophila mutant shibere that is paralyzed due
  to block in clathrin-coated pit uptake in neurons

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Lipid rafts

• Membrane microdomains enriched in glycolipids and
  cholesterol
• Some proteins are enriched in rafts, others
  excluded
• Form in the Golgi complex where they act as
  platforms for sorting and trafficking of
  apically-destined proteins
• Also thought to function at the PM in endocytosis
  and in organizing cell signaling pathways
• Controversial model

Simons and Ikonen (1997) Nature 387569
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How we get the textbook models examples from
recent literature

• Puertollano et al. (2001) Sorting of mannose
  6-phosphate receptors mediated by the GGAs.
  Science 2921712-1716
• Fölsch et al. (1999) A novel clathrin adaptor
  complex mediates basolateral targeting in
  polarized epithelial cells. Cell 99 189-198

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How are M6PR (which bind lysosomal hydrolases)
sorted into vesicles at the TGN?
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A-F. Fixed cells showing co-localization of GGA1
and M6PR G. Vesicular trafficking of YFP-GGA1 out
of the TGN in live cells H. Co-trafficking of
M6PR and GGA1 in live cells
Puertollano et al. 2001
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(Movie)
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• A dominant negative GGA1causes selective
  inhibition of M6PR exit from the TGN without
  affecting other transport processes out of the
  TGN or TGN structure
• LAMP-1 lysosomal membrane protein
• Tac PM protein
• TGN38 TGN resident protein
• AP-1 another TGN-associated adaptor

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A novel clathrin adaptor complex mediates
basolateral targeting in polarized epithelial
cells Folsch et al. 1999 Cell 99189-198

• AP1 (TGN-gt endosomes) contains m1, which
  recognizes tyrosine-based sorting signals
• Expression of an isoform of m1, m1B, is confined
  to epithelial cells
• LLC-PK1 cells lack m1B and missort LDLR and TfnR
  to the apical surface
• Exogenously expressed m1B in LLC-PK1 cells
  redirects LDLR and TfnR to the basolateral
  surface