Lecture 8: Membrane Transport, Protein Sorting, And Cell Communication (Part II):

ER ¡V cis Golgi Vesicle Trafficking Network:

1) ER- Golgi Route:

a) Cargo is wrapped around by a COPII-coated transport vesicle

b) This vesicle will bud from the ER exit sites

c) In some cases:

i) Before the cargo departs ER, cargo receptors (transmembrane protein) at ER membrane are bound by some of the cargo proteins, which act as the exiting signal of the vesicle

d) In other cases:

i) There are no exit signals for the departure of cargo

ii) These cargos package at a lower rate or have high concentration in ER0

e) Cargo proteins that are not folded properly are retained in the ER by chaperones until their degradation by proteasomes

f) After budding out of ER, the vesicles lose their COPII coat and fuse together to form tubular clusters

i) This fusion is promoted by the v-SNAREs and t-SNAREs of the vesicles

g) Tubular clusters move along microtubules and finally fuse with the cis Golgi

2) Golgi ¡V ER Route (Retrival Transport):

a) COPI-coated transport vesicles bud off the vesicular tubular clusters

i) Serves as a recycling mechanism to get proteins (i.e. BiP) back into the ER

ii) Depends on ER Retrieval Signal

b) Retrieval signals:

i) Usually located at the C-terminal end of a protein

ii) For ER membrane proteins, it is usually KKXX (Lys-Lys-X-X)

(1) KKXX sequence (Lys-Lys-X-X) on KDEL receptors (membrane protein) are there to bind the COPI coat

iii) For soluble ER resident proteins, it is usually KDEL (Lys-Asp-Glu-Leu or something similar)

(1) KDEL receptors (multi-pass transmembrane proteins) bind to KDEL sequence of the protein, allowing it to be sorted to the COPI-coated vesicles

(2) Removal of KDEL from BiP (by artificial means) result in (slow) net-secretion of BiP from the cell

(3) Addition of KDEL to a protein normally secreted by ER result in recycling of that protein back into ER0

3) Regulation of Proteins:

a) KDEL receptors have high affinity for KDEL in vesicular tubular clusters and golgi

b) KDEL receptors have low affinity for KDEL in ER (inducing its release in ER)

c) There is a pH gradient from ER (neutral) to Golgi (acidic)

i) This pH difference is regulated by V-type ATPases (proton pumps)

ii) The pH effect plays a crucial role in KDEL receptor binding:

(1) Low pH, high affinity

(2) Neutral pH, low affinity

d) Not all proteins have retrieval signals

e) Some Golgi enzymes cycle between ER and Golgi

i) They randomly enter COP1-coated transport vesicles going back to ER, but at a slow rate

f) Some ER resident proteins are retained at high concentrations in ER

i) Can be a result of the proteins forming complexes that are too large to enter transport vesicles

ii) At this concentration, low-affinity interactions are sufficient to get most of the proteins stuck in large complexes

4) Vesicle Trafficking in Golgi:

a) Vesicles from ER enter the cis Golgi network (CGN).

b) The materials that the cis network receives can be transported to the medial compartment where is can be further passed along to the trans Golgi network where they can be exported to other places in the cell

i) Trans Golgi network is the major branch point of the vesicle trafficking and proteins can be sorted into many different vesicles

ii) Vesicles for plasma membrane fuse with plasma membrane

(1) Can occur in two ways:

(a) Constitutive secretion: Immediate release and fusion with plasma membrane

(b) Regulated secretion: Like above, but waits for a signal first (usually Ca²⁺)

iii) Vesicles for lysosome (usually for degradation of macromolecules):

(1) First transported to late endosome and then to lysosome

(2) Lysosome uses hydrolytic enzymes that require low pH to function properly

(3) Pathway of entry for macromolecules from extracellular fluid involves:

(a) Endocytosis to early endosomes

(b) Recycling of some of these molecules back to the plasma membrane

(c) Those that are not recycled are transported to late endosomes

(i) Late endosomes have a slightly acidic pH (6) and degrades some macromolecules

(ii) When the late endosomes¡¦ pH are low enough, they form mature lydosomes

(4) Production of lysosomal hydrolase enzyme:

(a) Goes from ER è Golgi è Lysosome

(b) The precursor (N-linked Oligosaccharide) is transported from ER to cis Golgi network where a mannose-6-phosphate group was added to it

(c) The modified protein is then recognized by the Golgi enzyme GlcNAc phosphotransferase

(d) GlcNAc phosphotransferase adds GlcNAc-phosphate to mannose and later removes the GlcNAc leaving only M6P

(5) Regulation of Lysosome Hydrolase:

(a) Trans Golgi network has M6P receptors that bind to hydrolases with M6P

(b) These receptors bind lysosomal hydrolases on luminal side

(c) M6P receptors are sensitive to pH difference between TGN and late endosomes and would release cargo as a result of that

(d) Phosphatase enzyme removes phosphate from M6P to prevent rebinding

(e) M6P receptors are then recycled back to Golgi

c) Both the cis and trans systems are crucial and the flow of proteins in these networks are reversible

d) Each of the three compartments in Golgi (cis, medial, and trans) process N-linked oligosaccharides

i) Cis removes Mannose

ii) Medial removes mannose and adds GlcNAc

iii) Trans adds galactose and NANA

e) The transportation of proteins through Golgi can be explained by two models:

i) Vesicular transport model:

(1) Transport vesicles move from one Golgi compartment to next

(2) Golgi enzymes are held in place within each cisternae

(3) Different enzymes are in different parts of Golgi

(4) Retrograde transport resicles are there to recycle escaped ER/Golgi proteins

(5) Golgi is viewed as a static structure

ii) Cisternal maturation model:

(1) The Golgi cisterna are formed from the merging/clustering of vesicular tubular:

(a) Vesicular tubutlar è Cis cisterna è Medial cisterna è Trans cisterna è Molecular export

(2) Golgi is viewed as a dynamic structure

iii) Transport may very well occur by a combination of the two mechanisms where some cargo move rapidly by transported vesicles while some others move more slowly through cisternal maturation

f) Golgi structure depends on:

i) Microtubule cytoskeleton (holds Golgi together)

ii) Cytoplasmic Golgi matrix proteins (form scaffold)

iii) Matrix protein

(1) Forms long tethers that keep vesicles close to Golgi