pages 701 703, 705 709, 711 724

1
Chapter 12 13
Lecture 34 pages 701 - 703, 705 - 709, 711 - 724
Endoplasmic reticulum post-translational events,
lipid biosynthesis Vesicular Traffic
2
Nascent polypeptides enter the ER lumen
unfolded. Chaperone called BiP in the lumen
associates with hydrophobic surfaces to prevent
aggregation and to allow for proper folding. For
example, suppose a hydrophobic domain near the
N-terminus is suppose to associate with a
hydrophobic domain near the C-terminus of the
mature polypeptide. In the absence of
chaperones, the N-terminus could end-up
associating incorrectly with a hydrophobic domain
located elsewhere.
3
Protein disulfide isomerase (PDI) catalyzes
cycles of disulfide bond formation and breakage
until the correct disulfide bonds are formed.
Without PDI, incorrect disulfide bonds might form
and trap the protein in the wrong conformation.
4
N-linked glycosylation of ER proteins. Most of
the glycosylation associated with proteins
outside the cell begins in the ER.
A large preformed oligosaccharide is transferred
from a lipid called dolichol to the side-chain of
asparagine. Because the covalent linkage is to
the nitrogen of the asparagine side-chain, this
is called N-linked glycosylation. In the ER,
N-linked oligosaccharides are modified and the
modifications are used as signals to distinguish
properly folded from unfolded proteins.
5
GPI anchored integral membrane proteins are
generated in the ER.
6
Phospholipids are synthesized in the cytoplasmic
leaflet of the ER.
7
Phospholipid translocators flip-flop the
phospholipids.
8

• Transfer of lipids to other organelles.
• Most lipids for other organelles are synthesized
  at the ER.
• Lateral diffusion will supply the nuclear
  membrane.
• Vesicular transport will supply organelles in the
  secretory pathway and lysosomes (vesicular
  transport will be described soon)
• Phospholipid exchange proteins deliver
  phospholipids to the mitochondria, chloroplasts
  and peroxisomes.

9
Once proteins that dont normally reside in the
ER are properly folded, they are transported to
the golgi apparatus.
10
Vesicular transport delivers components between
compartments in the biosynthetic-secretory and
endocytic pathways.
Note how the cytoplasmic domain of my
hypothetical protein remains in contact with the
cytoplasm. Vesicular transport maintains the
membrane orientation.
11
Vesicular transport is performed by various
proteins that must do 3 things 1. Form the
vesicle with the correct cargo. 2. Target the
vesicle to the correct destination. 3. Fuse the
vesicle to the target membrane.
12
Three coat proteins drive vesicle formation at
various locations in the cell.
13
Clathrin associates via adaptins with receptors
in the donor membrane. The receptors bind
specific cargo. The clathrin assembles into a
cage that encapsulates a region of membrane.
Then dynamin causes the membrane to pinch off
forming a vesicle.
14
TEM detection of clathrin cages forming at the
plasma membrane.
15
Insert figure 13-7 to show assembly
16

• Clathrin-dependent vesicle formation requires
  energy.
• GTP hydrolysis by dynamin accompanies pinching
  off.
• ATP hydrolysis by chaperone proteins (not shown)
  accompanies the dismantling of the clathrin coat
  - the chaperone protein in this case is changing
  the folding of the clathrin so the molecules
  dissociate from each other.

17
COPII vesicle formation is mediated by a
monomeric GTPase. A GEF in the donor membrane
interacts with the GTPase, Sar1, causing GDP/GTP
exchange. Sar1-GTP extends a fatty acid tail
that inserts into the membrane. COPII assembles
on the Sar1 to form a vesicle.
COPI vesicle formation involves a protein called
ARF that is analogous to Sar1.
18
Uncoating of the COPI and COPII vesicles occurs
when the G-protein (ARF or Sar1) hydrolyzes GTP
and retracts that fatty acid tail. This may not
require a GAP but is instead dictated by the rate
of hydrolysis intrinsic to the G-protein.
19
Targeting of the vesicles is achieved by
complementary sets of v-SNAREs and t-SNAREs.
20
Conformational changes in the v-SNARE/t-SNARE
complex appear to drive membrane fusion without
ATP or GTP hydrolysis.
21
After membrane fusion, ATP hydrolysis is used to
pry apart the v-SNARE/t-SNARE complex.
22
Comparison of Clathrin and COP-dependent
formation of transport vesicles.