Endosomes: Protein sorting along the endocytic pathway

1
Endosomes Protein sorting along the endocytic
pathway
Tuesday Jan. 17, 2006 Sandra Schmid slschmid_at_scrip
ps.edu Phone [phone]
2
Endosomes were discovered in early 80s
Cell (March 1983) 32931-940 Penetration of
semliki forest virus from acidic prelysosomal
vacuoles Mark Marsh, Eva Bolzau and Ari Helenius
To identify and characterize the intracellular
site from which the penetration of Semliki Forest
virus (SFV) to the cytosolic compartment of the
host cell occurs, we determined the time course
and temperature dependence of nucleocapsid
uncoating and infection in BHK-21 cells. At 37C
the genome release to the cytosol was detected
within 57 min after virus endocytosis, whereas
delivery of the virus particles to secondary
lysosomes occurred within 1520 min. At
temperatures of 1520C virus particles were
internalized by endocytosis, but they were not
delivered to the secondary lysosomes.
Nevertheless, at 20C nucleocapsid uncoating and
infection occurred, indicating that secondary
lysosomes are not required for SFV penetration.
We conclude that the penetration reaction occurs
in prelysosomal endocytic vacuoles (endosomes).
As SFV penetration by membrane fusion requires a
pH lt 6 and the presence of cholesterol in the
target membrane, the data indicate that endosomes
are acidic and contain cholesterol.
3
Structurally pleiomorphic endosomes are
identified by endocytic content
A Quantitative Analysis of the Endocytic Pathway
in Baby Hamster Kidney Cells Gareth Griffiths,
Ruth Back, and Mark Marsh 1989 JCB 1092703
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Cell, Vol. 37, 195-204, May 1984 Intracellular
Receptor Sorting during EndocytosisComparative
lmmunoelectron Microscopy of Multiple Receptors
in Rat Liver Hans J. Geuze et al.
Fig. 8/9 IGA-R, 5 nm gold, ASGP-R, 8 nm gold
Sinusoidal PM and coated pits labeled with both
receptors
Fig. 10/11. IGA-R. 8 nm gold ASGP-R, 5 nm gold.
Receptors segregated to distinct endosome
tubules
Figure 12/13/14. IgAR segregated into
transcytotic transport vesicles for delivery to
basolateral membrane/ bile canaliculus.
Fig. 15. ASF is delivered to electron-dense
lysosomes and IgA to the transfer vesicle.
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Membranes of Sorting Organelles Display Lateral
Heterogeneity in Receptor Distribution Hans J.
Geuze, Jan W. Slot, and Alan L. Schwartz 1987
JCB 1041715
Recycling receptors are enriched in tubular vs
vacuolar portions of endosomes
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Characterization of Endocytic Compartments Using
the Horseradish Peroxidase-Diaminobenzidine
Density Shift Technique Richard S. Ajioka and
Jerry Kaplan 1987 JCB 10477-85
-DAB
DAB
EGF and LDL are present in HRP-Tfn-containing
endosomes 10 min after internalization
Percoll gradient fractionation and DAB-density
shift of HRP-Tfn-containing endosomes
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The endocytic pathway Sorting membrane traffic
from the cell surface and the TGN
t1/2 2 min
t1/2 2 min
t1/2 12 min
t1/2 2-3 min
t1/2 8 min
t1/2 15-30 min
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Immuno-EM and subcellular fractionation
studies establish that early/sorting endosomes
function to sort endocytosed proteins
WHY?
Membrane recycling Protein turnover Selective
delivery to specific subcellular
compartments Transcytosis/membrane polarity
WHERE?
Recycling endosomes Multivesicular
Bodies Transcytotic vesicles
HOW?
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General Principles Sorting along the endocytic
pathway involves 1) the establishment and
maintenance of endosomal subdomains 2)
selective segregation of cargo molecules into
these subdomains
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Two sites of protein sorting along the endocytic
pathway Early endosomes and Late endosomes
Carrier vesicle budding fusion Or Early-Late
Endosomal Maturation ????
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Sorting mechanisms that operate along the
endocytic pathway

• Geometry-based sorting
• pH-dependent receptor-ligand dissociation leaves
  receptors in membrane and ligands in solution
• tubular vs. vacuolar partitioning

• Physical property-based sorting
• lipid rafts, lysobisphosphatidic acid and
  cholesterol
• long, saturated acyl chain containing lipid

• Coat Protein-mediated, signal-dependent sorting
• Clathrin, AP1, AP2, AP3, AP4?, coatomer, ESCRT
  complex

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Sorting mechanisms that operate along the
endocytic pathway

• Geometry-based sorting
• pH-dependent receptor-ligand dissociation leaves
  receptors in membrane and ligands in solution
• tubular vs. vacuolar partitioning

• Physical property-based sorting
• lipid rafts, lysobisphosphatidic acid and
  cholesterol
• long, saturated acyl chain containing lipid

• Coat Protein-mediated, signal-dependent sorting
• Clathrin, AP1, AP2, AP3, AP4?, coatomer, ESCRT
  complex

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Tubular-vesicular Morphology of the Early
Endosome Creates Protein-sorting Domains
Membrane/volume tubules gtgt vacuoles
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Endosomal pH gradient and pH-sensitive
receptor-ligand interactions creates
spatio-temporal control of sorting along the
endocytic pathway
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Membrane Economics Duncan and Pratten (1977) J.
Theoretical Biol. 66727
Early endosomes
Vesicle of radius r Surface area 4Pr2 Volume
4/3Pr3 Consequently A sphere with double the
volume has only 1.6X the surface area
VTCs
MVB
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Iterative Sorting based only on geometry
Hypothetical sorting endosome
16 fusion events 1.6 fold excess
membranevolume All excess membrane in 50 nm
tubules Volume in vacuolartubular portion
51 Assuming tubules recycle, vacuoles are
delivered to lysosomes then 20 of volume will be
missorted/recycled Repeat 3 times (.2x.2x.2),
then lt1 of ligands are missorted
But what about the receptors? Over half the
membrane is in the vacuolar portion of the
endosome!
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Sorting mechanisms that operate along the
endocytic pathway

• Geometry-based sorting
• pH-dependent receptor-ligand dissociation leaves
  receptors in membrane and ligands in solution
• tubular vs. vacuolar partitioning

• Physical property-based sorting
• lipid rafts, lysobisphosphatidic acid and
  cholesterol
• long, saturated acyl chain containing lipid

• Coat Protein-mediated, signal-dependent sorting
• Clathrin, AP1, AP2, AP3, AP4?, coatomer, ESCRT
  complex

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Protein sorting by curvature/domain sensing
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Sorting based on lipid microdomains
Fred Maxfield Mukherjee et al. 1999 J. Cell Biol.
1441271
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Domain-based Segregation in the Sorting Endosomes
A working Hypothesis
Fred Maxfield
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Sorting mechanisms that operate along the
endocytic pathway

• Geometry-based sorting
• pH-dependent receptor-ligand dissociation leaves
  receptors in membrane and ligands in solution
• tubular vs. vacuolar partitioning

• Physical property-based sorting
• lipid rafts, lysobisphosphatidic acid and
  cholesterol
• long, saturated acyl chain containing lipid

• Coat Protein-mediated, signal-dependent sorting
• Clathrin, AP1, AP2, AP3, AP4?, coatomer, ESCRT
  complex

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Coat proteins can aid receptor recyling
A Novel Class of Clathrin-coated Vesicles Budding
from Endosomes Willem Stoorvogel, Viola Oorschot,
and Hans J. Geuze JCB 1996 13221
A. Tfn-HRP containing endosomes fixed with DAB
and stained with anti-Cl gold. Large buds
coming, small buds going?
B. Tfn-HRP containing endosomes fixed with DAB.
Small buds label with anti-Cl gold, larger buds
are not labeled

• Arrowheads point to flat
• DAB-negative clathrin lattices on the PM.

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Hrs/Stam, clathrin, and ESCRT complexes in MVB
formation
Annexin II role in tubule formation
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Protein sorting along the endocytic pathway the
multivesicular body
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Hrs/Stam recognizes monoUbiquitin for sorting
into lumenal vesicles
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The ESCRT machinery and sorting into MVB
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How are sorting domains of the endosomal
compartments established?
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An important role for lipids in establishing
membrane subdomains and in protein sorting
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Rapid interconversion of PIP species helps
generate/identify new functional membrane domains
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PIPs as Compartment-Specific Tags
Plasma Membrane
PI4,5P2
PI3P
PI4P
Endosomal
System
Golgi
Complex
PI3,5P2
ER
Lysosome/
Vacuole
Emr Lab Chris Burd Chris Stefan Jon Audhya
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PI3P
FYVE
DsRed
PI(4,5)P2
PH
GFP
vacuole
CMAC
Emr Lab Chris Stefan Jon Audhya
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Lipid composition and pH gradient are sufficient
to generate interlumenal invaginations In model
liposomes
H. Matsuo et al., Science 303, 531 -534
(2004)
Published by AAAS
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The endosomal compartment is organized
into functionally distinct microdomains
Miaczynska and Zerial (2002) Exp. Cell Res.
2728-14
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Rab4 and Rab5 effectors determine early endosomal
function

• Endosomal Rabs and their effectors
• set up positive feedback loops to create
  membrane domains
• recruit downstream rab to generating new sorting
  domain
• recruit docking and fusion factors for homotypic
  fusion

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The complex network of Rab5 regulators and
effectors involves positive feedback loops
designed to generate a local amplification of
active Rab5 and the clustered recruitment of Rab5
effectors on the early endosome membrane.
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Rab5/Rab4 domain interface is established by rab5
effectors
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SNX-family of PX domain-containing proteins
function in protein sorting along endocytic
pathway
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SNX-family of PX domain-containing proteins
function in protein sorting along endocytic
pathway
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Take Homes on Endosomal Sorting

• Rab proteins control domain organization within
  endosomes
• Rab effectors create positive and negative
  feedback loops (by controlling
• PI-kinases/phosphatases and rab GEF/GAP activity)
  to stabilize or destabilize endosomal domains
• Sorting machinery is targeted to endosomes
  through PI3P interactions and interactions with
  rabGTP
• Cargo can be sorted into domains by peripheral
  protein coats, geometry, preference for lipid
  environment or curvature, etc.