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The Secretory Pathway
Becky Dutch
Molecular and Cellular Biochemistry
1. ER - translation
2. ER- protein modifications
3. Discussion Section
4. Golgi apparatus
5. Vesicular transport
Lecture 3:
The Golgi apparatus
Reading: Alberts Chapter 13
Lodish Sections 17.7 and 17.8
Modification and Sorting
in the Golgi:
1. Addition, Processing of
carbohydrates - ER,
Golgi
2. Specific Proteolytic
cleavages - Golgi
3. Sorting to proper
destinations
. Lodish, Fig 17-13
Golgi Apparatus: collection of flattened,
membrane-bound cisternae.
Alberts 13-3
Proteins from ER enter at cis face, go through
different compartments - then leave TGN for their
final destinations.
Alberts 13-4
Cisternal Progression or vesicular transport?
-How do proteins
move from cis to
medial to trans?
-Originally thought
to be by transport
vesicles (COP1).
Alberts 13-14
Cisternal Progression/Maturation
Evidence - Biosynthetic aggregates (collagen precursors)
too large for vesicles traverse the Golgi stack. No
evidence for a megavesicle. Rate of aggregate movement
much slower than for normal proteins.
Model - cisternae “progress” or mature. Cis Golgi
becomes medial becomes trans. Golgi enzymes are
moved back by retrograde transport.
Vesicular Transport in Golgi
Evidence: Two distinct forms of COP1 vesicles those with KDEL receptor and little cargo, and those
with lots of cargo, no KDEL receptor. What is
this second population doing? Also, transport
for many proteins is faster than seen for aggregates.
Model: COP1 vesicles mediate transport from cis to
medial to trans.
Cisternal Progression and vesicular transport
Model: Both may
function in transport
“Slow Track” cisternal progression
“Fast Track” - COP1
vesicles
Pelham and Rothman,
Cell 102: 713-719.
Alberts 13-14
Processing glycoproteins in Golgi
Cis, medial and trans compartments
have different enzymes
High mannose form - reactions
1 or 2 do not occur - Man8(GlcNAc2)
or Man5(GlcNac2) - do not get
further additions in Golgi
Complex carbohydrates - additions
in the Golgi
Lodish 17-38
Endo H digestion distinguishes complex from
high mannose
Alberts 13-11
How do nucleotide sugars get into the Golgi?
Antiporters located in the
Golgi membrane
One-for-one exchange keeps
the concentration of sugar
nucleotides constant
Lodish 17-33
Targeting proteins to the lysosome
Addition of P to Mannose 6 is needed for recognition
by the Mannose - 6 - P receptor
Signal sequence binds recognition site, catalytic site
is distinct
Lodish 17-39
Mannose - 6- phosphate pathway
Lodish 17-40
Lysosomal enzymes
phosphorylated
Bind M6P receptor
in TGN
Directs incorporation
into clathrin-coated
vesicle
Uncoating, fusion with
late endosome
Low pH of endosome
results in receptor
release - P removed
Receptor recycles,
enzyme goes to
lysosome
Disorders in lysosomal enzyme sorting
Inclusion cell disease (I cell disease) - rare disorder in which
almost all hydrolytic enzymes are missing from lysosome
I cell disease - single gene, recessive defect
Alberts 13-23
Hydolases found
in blood instead
Defective or missing
GlcNAc-phosphotransferase
No P, no binding
M6P receptors
Some cell types
(heptacytes) still sort
to lysosome - must be
an M6P independent
pathway
Retention of resident Golgi Proteins
All made in ER - carbohydrate modifying enzymes have
similar structure: short N-terminal domain that faces
cytosol (so Type II), single TM a helix, large C-terminal
domain facing Golgi lumen
Membrane spanning domain necessary and sufficient
for retention in the Golgi - mechanism of retention not
known. Not one single sequence of TM.
Lodish 17-21
Sorting into Vesicles
TGN to plasma membrane - two major types of vesicles:
secretory vesicles for regulated secretion and transport
vesicles for constitutive secretion
How do proteins get sorted to the correct vesicle? Common
mechanism seems to function for many regulated
secretory proteins (ACTH, insulin, trypsinogen) but don’t
share common sequence.
Hypothesis: selective protein aggregation. Many mammalian
cells have secretory vesicles with chromogranin B and
secrotogranin II - form aggregates in TGN conditions (pH
6.5, 1mM Ca)
Proteolytic processing in the TGN
Most secretory proteins, some plasma membrane proteins made as proproteins - must be proteolytically cleaved to
be active. Examples include insulin, glucagon, HIV env
Some of these
cleavage events
occur in secretory
vesicles as they
leave TGN
Ab to proinsulin
Lodish 17-41
Insulin - only
mature vesicles
show cleaved form
Ab to insulin
Some proteins have single cut site
Furin - endoprotease. In all mammalian cells. In
the Kex2 family of endoproteases.
Lodish 17-42a
Some secreted proteins undergo multiple cleavages
Proinsulin - regulated secretion.
PC2 and PC3 endoproteases - also in Kex2 family. Only found
in cells with regulated secretion.
Localized to secretory vesicles.
Three cleavage events needed
to form insulin.
Lodish 17-42b
Apical/Basolateral Sorting
Polarized epithelial cells - plasma membrane has two domains:
apical and basolateral. Separated by tight junctions.
Proteins sorted to
one or other membrane
One way: sorting in TGN
- distinct vesicles go to
the two surfaces
Examples
Influenza HA - apical
VSV G - basolateral
GPI-linked - apical
(in endothelial;
basolateral in thyroid)
Lodish 17-43
No unique sequence for
targetting
Apical/Basolateral Sorting II
Hepatocytes - another sorting mechanism.
All proteins targetted
to basolateral membrane
Both types endocytosed
Basolateral recycled to
basolateral membrane
Apical move to apical process called
transcytosis
Lodish 17-43
Next lecture:
Vesicular Transport
. Lodish, Fig 17-13