MBB231 - Lecture 12 : Vesicular Transport

what is protein targeting

proteins synthesized in the ER have to be directed to various locations

• each protein contains a specific tag that targets a transport vesicle → takes it to the correct location

why is lysosomal targeting the most established organelle targeting strategy

• automatically collects molecules from outside of the cell

• features a unique acidic environment that makes drug delivery simple → acidic environment can activate designed drugs

what are protein residence

• proteins that are kept within an organelle / compartment of an organelle

what are the two mechanisms in keeping proteins in an organelle or specific compartment

retention - resident molecules are excluded from transport vesicles (induces local containment)

retrieval - tags are used to return proteins to it’s proper location

what is the function of the proteins that coat the vesicle

• curve the membrane to be able to form the vesicle

• select components that will be carried in the vesicle

the vesicle coat is composed of what layers and what do they do

outer layer - acts as a cage around the vesicle

inner layer - acts as an adaptor between the outer layer and the lipid bilayer

how does the inner layer select cargo proteins

through affinity interactions with the cytoplasmic domain of the transmembrane proteins

how are soluble proteins of the lumen selected

cargo receptor acts as a middleman:

• one end of the cargo receptor grabs the soluble proteins in the lumen

• other end of the receptor sticks out of the membrane - inner coat layer can grab it

what are the 3 main types of coat proteins and what is their function

COPII - ER → cis-Golgi

COPI - golgi → ER & Golgi → cisternae

Clathrin - plasma membrane → endosome

what is the function of the COPII-coated vesicle

moves material from ER → to the ER-golgi intermediate compartment + Golgi (forward)

what is the function of the COPI-coated vesicle

• moves materials from the ER-golgi intermediate compartment + golgi back to the ER (backward)

• or from trans golgi → cis golgi cisternae

what is the function of the clathrin-coated vesicles

• moves materials from the plasma membrane backwards

• TGN → endosomes / lysosomes / plant vacuoles

how is a vesicle formed from a donor membrane

budding initiated: initiated by recruitment of a small GTP binding protein to a patch of donor membrane

budding: driven by the formation of a proteinaceous vesicle coat

gathering proteins: interactions between the vesicle coat and the transmembrane proteins gather the selected cargo proteins

fusion: integral membrane proteins in the budding vesicles (such as v-SNARES) are important for fusing with the correct target membrane

• after the vesicle is released + sheds its coat → fuses with the target membrane in a process that involves interaction of SNARE proteins

budding is initiated by the recruitment of what small GTP binding protein to the patch donor membrane

SAR1-GDP

how does a cell start building a COPII vesicle

• small protein Sar1 floats near the ER membrane → switched off as it is carrying GDP

• helper protein Sec12 (embedded in the membrane) swaps out the GDP for a GTP molecules → Sar1 is switched on - changes conformation

• changing conformations exposes a hydrophobic N-terminal alpha helix on Sar1 → interacts with the membrane → locking the protein in place with the membrane

• results in the bend of the membrane → into a bud

what coat proteins does SAR1-GTP recruit and why

recruits coat proteins: Sec 23 & Sec 24

• these coated proteins have a curved conformation → bends the membrane

• Sec24 also acts as an adaptor protein - helps in recruiting specific proteins and packs them into vesicles

what does Sec13 & Sec31 do

they bind together to form the outer layer

in transit why must the protein coat disassemble

it allows for membrane proteins (i.e. v-SNARES) to target the vesicle to the proper membrane

how does the protein coat disassemble

hydrolysis of GTP - forming SAR1-GDP (inactive) → no longer associates with the membrane and the vesicle coat falls of

what happens if you can’t hydrolyze GTP

Sar1 remains in the GTP activate state → vesicle coat remains as uncoating is blocked

the movement of vesicles is mediated by what

microtubules

the initial contact between a vesicle and the target membrane involves what kind of proteins

tethering proteins

what are the two kinds of tether proteins

• rod shaped fibrous proteins → forms long bridges

• multi protein complexes → holds two membranes close together

how do Rab GTPases help in specificity and targeting

there are 60 different types of Rab proteins → certain regions have their own specific Rab proteins - ensures a vesicle attaches to the targeted membrane

how does the process of specificity occur with Rab proteins

• Rab protein becomes active when bound to GTP → forms Rab-GTP

• Rab-GTP recruits the cytosolic tether proteins to the membrane surface

• tether proteins reach out from target membrane and catch incoming vesicles

• tether pulls the vesicle close enough to the target membrane → snare proteins ( v-SNARE / t-SNARE ) lock together and fuses the two membranes

what is the SNARE hypothesis

that the sorting and targeting of vesicles involves two families of SNARE → v-SNARE (vesicle SNAP receptor) + t-SNARE (target SNAP receptor)

how does the fusion of the vesicle and plasma membrane occur

• v-SNARE on vesicle twists tightly together with t-SNARE on the plasma membrane → pulls the two membranes close together

membrane merging (living cells): influx of calcium ions triggers the two membrane’s outer layers to press together → start to blend into one barrier

fusion pore opening: merged membrane forms a fusion pore → allows the vesicle to expel contents outside of the cell

what’s the difference between membrane fusion in vitro and vivo

vitro (in glass) - interaction of v-SNARE / t-SNARE is strong enough to cause membrane fusion

vivo (life) - rise of calcium concentration initiates membrane fusion

how does dissociation occur after membrane fusion

requires NSF (N-ethylmaleimide-sensitive factor) and SNAPs (soluble NSF attachment proteins) to pray apart the SNAREs → using the energy from ATP hydrolysis (ATP → ADP + Pi)

how are soluble ER proteins returned to it’s original location (ER)

• tagged with KDEL sequence (Lys-Asp-Glu-Leu) at the c-terminus

• if the protein drifts into the golgi → receptors in the golgi recognize the sequence → pack the protein into a transport vesicle back to the ER

how are transmembrane ER proteins returned to it’s original location (ER membrane)

• is tagged with KKXX sequence (Lys-Lys-any amino acid-any amino acid) - located on the part of the protein that sticks out into the cytosol

• tag binds directly to vesicle coat proteins → pinches off the membrane and carries the protein back in a vesicle → to the ER

COPI coat is made up of what

a complex of 7 proteins → cotamer

what initiates vesicle formation and disassembly in COPI

vesicle formation initiated by: small GTPase - Arf1

coat disassembly: Arf1-GTP → Arf1-GDP through hydrolysis

how does the retrograde transport of soluble ER resident proteins work

• soluble ER proteins with a KDEL sequence tag can leak out of the ER into COPII-coated vesicle → heads to golgi

• golgi lumen is slightly more acidic than the ER → causes membrane bound KDEL receptors to bind tightly to the KDEL tag on proteins

• KDEP receptor cluster into COPI-coated recycling vesicles → buds off and travels backward to the ER

• once the vesicle fuses with the rough ER - high pH lowers the receptors affinity → release the resident protein to it’s proper location

→ pH acts as a molecular switch:

Low pH in golgi → activates receptor binding to obtain stray proteins

high pH in the ER → deactivates binding to release the protein to its original location