Lecture 12 Vesicular Transport: the ER and the Golgi Apparatus:

Trafficking.

Lipid synthesis occurs in the cytosolic layer of the smooth ER using transmembrane
enzymes that are inserted into the bilayer. Lipids must be made inside the membrane
because they are so hydrophobic. Most of the components use to make a lipid is on one
layer. So technically only one side should get new lipids. But there are transmembrane
proteins called flippases which translocates the cytoplasmic leaflet to the luminal
leaflet. This allows for a lipid bilayer

Proteins destined for the Golgi, endosomes, lysosomes and cell surface first enter the
ER. Proteins move between these compartments by membrane traffic of vesicles.

Membrane Traffic: how does it work on a basic basis

1. Sort (what is going to be put inside the vesicle)
2. Budding ( making the vesicle to put the molecules in)
3. Targeting ( vesicle have to go to a specific area)
4. Fusion ( they have to fuse with the other membrane) the luminal phospholipids
and cytosolic phospholipids will be placed exactly as they were and the solunle
content will be delivered to the next cell

Vesicle trafficking from the ER to the Golgi:

Making the vesicle: a protein coat forms (COPII) on the cytosolic part of the
membrane as it builds up it bends the membrane, pinches off and makes the vesicle. A
switch devises (protein Sar1) it works with Sec proteins (yeast cells found in human
cells too) in order to build the coat.

Sar 1 protein:
- It is a member of the superfamily of trafficking and signalling
- It requires two protein partners the GAP protein and the EF (exchange factor) ( it
is a membrane protein)
- They are GTP binding proteins which need a GDP exchange factor that removes
the diphosphate and replaces it with a GTP ( as a whole)
- It needs a GTPase Activating Protein (GAP) converts GTP to GDP and a phosphate
- GTP= ON GDP= OFF
- The partners are one of the Sec proteins as the Exchange Factor is a Sec12
protein the Gap protein is Sec 23/24
Leaving the ER: bending the membrane
The alberts way 1:

When Sar1 is bound to GDP it is inactive but soluble so it cab

float around in the cytoplasm. When it gets replaced with GTP,
it flips the amphipathic helix and then te hydrophobic surface
will collide with the first hydrophobic surface it comes across
which is the hydrophobic area of the ER membrane.

Alberts way 2:
Sec23 and 24 are sitting on the membrane as a
membrane protein. Sec 24 doesn't directly attach to
Sar1, it attaches to the receptor molecules (cargo
receptor) inside the membrane of the ER which bind
onto proteins that you want to incorporate into the
vesicles. They build up a coat of proteins by collect cargo.
At this stage proteins are built up. But other Sec23/24
complexes are acting at the same time not just this one.

Alberts way 3:
As the Sec23/24 complexes build up, by binding with
eachother. The coat itself (Sec 13/31) begins to form by bending
the membrane from a cytosolic side until it bends so much that it
pinches off. Now, the Sec23/24 proteins are activated, it gets the
Sar1 protein to hydrolyses its GTP, flipping back to its
conformation. As everything is built onto the Sar1 as that flips
back into shape everything ( vesicle) falls off the membrane, this
causes the vesicle to float in the cytoplasm to the Golgi.

The alberts way 4:

That whole structure is called the COP-II coated vesicle.
There are a family of proteins used for targeting, 2 of them:

First family Rab: tethering

Second family: SNARE, docking and fusion

Rab proteins:
- There is a different member of this Rab family for every different membrane
traffic, different Rabs found on different membranes
- They can attach to membranes, by using a lipid anchor but ARE NOT
conventional transmembrane protein.

Tethering proteins help the protein get to the right areas of the membrane. They
interact with the tether proteins (dark green). They are also situated on the cytoplasmic
membrane. Then the SNARE proteins (second family of proteins which help with
docking and fusion) take over they get the vesicle to fuse with the membrane by
bringing it very close to the membrane. Membranes are hard to fuse together as they
have a net negative charge, these proteins force them together.

Snare proteins (extra); mediate membrane fusion:

- Snare proteins on the vesicles are called v-SNARES and they interact with the
snares on the target membrane called t-SNARES. There are different SNARE
proteins for different stages of traffic. The a-helical domains twist around one
another, driving fusion of the two lipid bilayers.
Sorting problems:

There are different coats for Anterograde and

Retrograde transport:
Vesicle transport from ER to Golgi (Anterograde):
COPII coated vesicles
From Golgi to ER (retrograde): COPI coated vesicles

Retention in the ER: reside in the ER

- Some ER proteins are soluble but stay in the ER, so
sometimes they get incorporated into the vesicles, but
they shouldn't normally
- They have a C-terminal tag (ER retention signal) called
KDEL. If the KDEL protein escapes then the receptor
binds to receptors in the Golgi, and it returns to the ER

The Golgi apparatus: vesicles enter from the cis-Golgi network

(facing the nucleus that looks like the ER, pass through the
cisternae and then exit by the trans-Golgi network.

Functions:
-less smaller in area than the ER
- it glycosylates proteins/ molecules
- it sorts proteins to the plasma membrane, lysosomes or
sends them back to the ER

Their appearance depends on the cell type.