4. From the ER to the Golgi Apparatus

Types of transport mechanisms

gated transport

transmembrane transport

vesicle transport

describe gated transport

transport through relatively large pore, but entry and exit controlled

nucleus/nuclear pore

describe transmembrane transport

membrane bound protein translocators move proteins across the membrane

ER, mitochondria, chloroplast

describe vesicular transport

membrane enclosed vesicles bud and fuse to move proteins from compartment to compartment

ER to Golgi and beyond, endocytosis

__ have been incredibly useful in understanding the secretory pathway

yeast mutants

what does the golgi apparatus do

modifies the oligosaccharides or proteins delivered from the RER

the golgi apparatus is a stack of __

flattened membrane compartments, each with specific enzymatic functions

• functionally equivalent compartments are interconnected by a tubular network

the size and number of ___ within the golgi apparatus varies with ___

stacks; cell type

• cells that export lots of proteins (pancreatic cells) have lots of golgi

five distinct compartments of the golgi

1. Cis Golgi Network (CIS) - site of delivery from ER

2. Cis Golgi - next region nearest the CGN

3. Medial Golgi - the central region

4. Trans Golgi - the distal region

5. Trans Golgi Network (TGN) - site of sorting and exit of vesicles from Golgi network

function of cis golgi network

site of delivery from ER

function of trans golgi network

site of sorting and exit of vesicles from Golgi network

after entering the golgi, proteins may ___, ___, or ___

return to the ER, remain in Golgi, be secreted from cell

the organization of the golgi at the ___ provides a mean for vesicles to move in a directed fashion via ___

microtubule organizing center; motor proteins

when proteins reach the cis-golgi, some of their ____ will be trimmed. proteins can be extracted from cells, and treated experimentally with endoglycosidase-D. the glycosidase will remove the whole ___ but only if ___

mannoses on their “sugar tree”; oligosaccharide tree; the mannoses had been trimmed in the golgi

proteins on the cytoplasmic face of the vesicles regulate ___

budding and fusion; 1 - Budding

the mechanical process of membrane budding is driven by a complex of ___ that assembles on the ___ they coat the vesicle surface so they are called ___

cytoplasmic proteins; outside (cytoplasmic face) of budding vesicles; coat proteins

different vesicle types have different ___

• __ - coats vesicles moving from ER to Golgi

• __ - coats vesicles moving Golgi to ER and from more trans Golgi to more cis Golgi

• __ - coats vesicles moving from TGN to endosomes, lysosomes and from surface to endosomes, lysosomes’

coat proteins

• COPII

• COPI

• Clathrin

COPII

coats vesicles moving from ER to Golgi

COPI

coats vesicles moving Golgi to ER and from more trans Golgi to more cis Golgi

Clathrin

coats vesicles moving from TGN to endosomes, lysosomes and from surface to endosomes, lysosomes

different coated vesicles serve ___

specific transport steps

how can different coated vesicles be distinguished?

morphologically

a class of small G proteins regulates ___

• these cytoplasmic G proteins have a lipid anchor that is ___ when GTP bound, or ___ when GDP bound, allowing them to ___

  • GTP bound → anchor __ → ___

  • GDP bound → anchor __ → ___

• different types of coat proteins use a different G protein:

  • COPII: __

  • COPI, Clathrin: __

budding

• exposed; hidden; reversibly associate with membranes

  • exposed → budding is initiated

  • hidden → no budding, or removal of coat

  • Sar1

  • Arf

non hydrolyzable analogs of GTP such as GTP-gammas trap ___

vesicles in the coated form

The vesicle cargo

• some soluble proteins seem to be ___

• other soluble proteins bind to ___. these transmembrane cargo receptors interact directly with coat proteins via their ___

• some membrane proteins can themselves ___

• thus, various signals for export are needed, the sugars or protein domains that allow soluble proteins to bind cargo receptors; the cytoplasmic domains of the cargo receptors or transmembrane proteins being packaged into vesicles by coat proteins

• carried along non-specifically by “bulk flow'“

• cargo receptors which concentrate them in vesicles (the cargo receptor ERGIC-53 is a lectin that binds the N-linked oligosaccharides of some cargo)

• directly interact with coat proteins via their cytoplasmic domains

the signals for export in cargo binding receptors or transmembrane cargo proteins themselves are ____

AA sequences that interact with coat proteins

BUDDING SUMMARY

• __ activated, __ begin to bud membrane

• Cargo loaded by bulkor by __ with coat proteins

• vesicles __

• COPII coat/Sar1 hydrolyze GTP, coat disassembles

• now need to ___

the coats disassembled after ___

• this means ___

• Sar1; COPII proteins

• direct or receptor mediated interaction

• bud off

• dock and fuse to target membranes

budding

• the coat does not play a role in targeting

docking and fusion are regulated by yet another ___

class of small G-proteins

• Rab family of small G-proteins

Rab family of small G-proteins

• GDP bound - __

• GTP bound state - __

• RabGTP mediates ___ of vesicles by binding the ___ in target membranes

• over __ different Rab family members! the specificity of vesicular trafficking pathways is in part controlled by specific __ used for different routes

• activated by __

• inactive, cytoplasmic

• active; tightly associated with membranes

• tethering and docking; Rab effector proteins

• 60; Rabs and effectors

• RabGEFs

transmembrane proteins called ___ control fusion of the vesicle to the target membrane

SNAREs

V-Snares in vesicle membrane are recognized by ___ on the target membrane (provides directionality between compartments)

T-SNARES

__ in vesicle membrane are recognized by T-SNAREs on the target membrane (provides directionality between compartments)

V-SNAREs

many unique T-SNARE and V-SNARE proteins exist and specific parings mediate __

specific membrane fusion events

together ___ controls the specificity and directionality of vesicular delivery and fusion

Rabs and SNARES

ER-Golgi vesicle trafficking

• __ initiates membrane budding process

• recruits ___ to drive budding

• receptor bound proteins, membrane proteins, and free proteins are recruited into the budding vesicle

• the vesicle ___

• the adaptor coat ___

• vesicle finds and docks its target membrane via ___; SNARE complexes drive ___

• __ has thus contributed to the CGN

• Active Sar1 G protein

• cytoplasmic COPII

• buds

• falls of

• Rab G-proteins, Rab effectors and SNARE proteins; fusion

• COPII vesicle transport

what controls exit from or return to the ER?

ER export signals

ER retrieval signals

ER export signals can ___

interact directly or indirectly with coat proteins. along with bulk flow, this moves proteins to the Golgi and beyonn

proteins that are supposed to function in the ER are ___

recycled back to the ER

• protein disulfide isomerase; catalyzes Cys-Cys disulfide bonds at ER membrane

• Chaperones that aid in folding; Bip, a soluble ER protein

• SRP receptor, an ER membrane protein

an ER retrieval signal for soluble proteins is ___

KDEL (Lys-Asp-Glu-Leu)

• the KDEL ER retention signal is at the C-terminus

• KDEL binds a KDL receptor in the Golgi membrane

• KDEL receptor interacts with COPI coat proteins on cytoplasmic side

(T/F) there are other ER retrieval signal sequences for membrane proteins

true

• these are not always at the c-terminus, and interact directly with COPI

KDEL receptor cycles between __ and __; differences in __ between the ER and later compartments may control binding and unbinding to receptors

ER and Golgi; pH

ER retrieval signals in soluble proteins (KDEL) interact with ___

transmembrane receptors that can interact with COPI coat proteins

transmembrane proteins can use other retrieval signals to directly interact with ___

COPI coat proteins to return proteins to the ER

How is the flow of material through the Golgi accomplished?

each golgi compartment (cis, medial, trans) has a unique enzyme composition to modify oligosaccharides

Vesicle Transport Model (flow of material through Golgi)

• vesicles bud from one ___ and fuse with the next to ___

• the golgi compartments themselves are relatively __

• golgi compartment; move cargo proteins and membrane forward from cis > medial > trans

• static, with each maintaining their specific processing enzymes

problems with the flow of material through the golgi

• while COPI vesicles, which are known to ___, have been found throughout the golgi complex, ___ have not

• some proteins such as pro-collagen fibers are ___ to be trafficked in coated vesicles, yet ___

• mediate retrograde (backward) movement; anterograde vesicles (COPII)

• too large; are known to be processed

Cisternal Maturation Model (flow of material through Golgi)

• a given ___ is not fixed, but matures, both spatially and enzymatically

• cargo-containg ___ from the ER fuse, generating a __, and cargo is subject to __ in that cisterna

• meanwhile another cis-golgi is forming behind the first, and the trans-golgi at the other end of the network is diminishing, as it delivers it membrane bound cargo onward. in this way, ___, becoming medial with respect to others and ultimately will become ___

• at the same time, ___ are mediating retrograde transport of enzymes from more trans- compartments to more cis- compartments. this means that a given compartment will be losing __ and gaining __; thus it “matures” from cis- to medial- to trans- golgi as it is moving __

• the cargo remains __ as the enzyme composition of that compartment changes, and the compartment is __

• golgi cisterna (compartment)

• COPII vesicles; cis-golgo; cis-golgi enzymes

• the original cis- compartment is moving in the trans- direction

• COPI vesicles; its cys- enzymes; trans- enzymes; through the complex

• in its original compartment; displaced toward the trans region