Lecture 16 Endoplasmic Reticulum And Golgi Apparatus

Question 1

Secreted proteins from ER to extracellular space

Answer 1

Synthesised by ribosomes that dock to ER
Mediated by signal sequence at N terminus
Proteins passed into ER lumen
Modified and passed onto Golgi where they are further modified
Finally they are transported out of the cell

Question 2

Some proteins are synthesed and folded correctly in ER lumen

Answer 2

New proteins from ER bound ribosomes fed into ER lumen.
Chaperones mediate folding (e.g. Nip binds hydrophobic regions preventing aggregation)
disulphide bonds are added by protein disulfide isomerase PDI

Oligomerisation occurs converting reduced protein to oxidised (native) protein. Disulfide bonds stabilise structure in secreted proteins

Question 3

Step 1: docking to ER

Answer 3

Involves a single peptide (SP) signal recognition particle (SRP)
and SRP receptor

Docked ribosomes synthesise membrane, lumenal or secretory proteins.

Ribosomes start reading mRNA in cytosol

SP is synthesised at NH2 terminus (first)

SP is recognised by SRP

Docks to SRP receptor in ER membrane

Transferred to Translocation channel
(Aka translocon)

Nascent protein synthesised through Translocation channel into ER lumen

Proteins need a channel that is aqueous as they are hydrophilic, cannot pass through membrane directly it’s hydrophobic

Question 4

Step 2: translocation through ER membrane (soluble excretory proteins)

Answer 4

Signal peptides are cleaved by signal peptidase releasing protein from translocon into ER lumen

Question 5

Step 2: translocation through ER membrane (membrane proteins)

Answer 5

(type 1) integral membrane proteins start or stop transfer sequences (transmembrane domain) are hydrophobic sequences that remain in the membrane

N terminal sequence is cleaved

Internal sequences become the transmembrane domains

Ribosome synthesises protein until it reaches the transmembrane domain and translocon dissociates

Internal “signal sequence” binds SRP which binds to SRP receptor, targets ribosome to translocon - it is not cleaved

Question 6

Proteins can be inserted into membranes in several ways

Answer 6

Type 1 - LDL receptor
e.g. influenza HA protein, insulin receptors and growth hormone receptor

Type 2 - Asialoglycoprotein receptor
E.g. transferrin receptor, Golgi galactosyltransferase and Golgi sialytransferase

Type 3 cytochrome P450

Type 4 G protein coupled receptors e.g. glucose transporters, voltage gated Ca2+ channels ABC small molecule pumps, CTFR (Cl-) channels, Sec61

Question 7

Type 1 single pass integral membrane protein

Answer 7

(see step 2 for membrane protein)

Single pass through membrane
Cleaved signal sequence at N terminus
N terminus on lumenal side
C terminus on cytosolic side

Question 8

Type 2 membrane protein synthesis

Answer 8

N terminus in cytosol and c terminus in lumen.
No cleaved signal sequence, instead an internal start transfer sequence.
If internal start transfer sequence is preceded by amino acid side chains it’s usually a type 2. This section prevents this part of the protein from entering the translocon and instead a hydrophobic signal sequence is inserted - high charged region excludes N terminus from passing through translocon

Question 9

Type 3 membrane protein synthesis

Answer 9

Looks like a type 1 but has no cleavable signal sequence instead has a run of charged aminos after it’s transmembrane sequence N terminal lumenal side and C terminal cytosolic side

Question 10

Type 4 membrane protein

Answer 10

No. Of internal transfer sequences determines no. Of transmembrane domains. Internal sequences bind SRP that binds to SRP receptors which targets ribosome on protein to translocon without cleavage.

Position of pos residues determines orientation of type 4
pos amino sequence pre transmembrane domain - n terminus cytosolic
Pos amino sequence post transmembrane domain n terminus lumenal

Odd no. Of loops leads to each end being on diff sides of the membrane

Question 11

Summary of soluble secretory protein synthesis in ER

Answer 11

Synthesised by ribosomes
N-terminal signal sequence
Recognised by SRP
sRP recognised by SRP receptor
Docks on translocon
Polypeptide translocated into ER
Signal sequence cleaved by signal peptidase
Folding aided by chaperones
Disulphide bonds formed
May oligomerise

Question 12

Summary of membrane protein synthesis in ER

Answer 12

Similar to soluble secretory protein synthesis
Some do and some don’t have N-terminal cleavable signal sequence
Have 1 or more internal hydrophobic sequence
Orientation determined/juxtaposed by positive residues

Question 13

Glycosylation

Answer 13

Proteins are glycosylated in ER - addition of polysaccharides.

Thought to protect proteins from degradation makes them more hydrophilic reducing aggregation and aiding folding

N-linked glycosylation (asparagine, Asn, N)

Starts in ER as protein is synthesised

Question 14

Smooth ER

Answer 14

Connected to rough ER

Exit site for transport vesicles (maybe?)

Synthesises lipids and steroids

Abundant in cells that metabolise lipids

E.g. Leydig cells in testes- extensive smooth ER to produce testosterone from cholesteron

Question 15

Function of ER summary

Answer 15

Site for membrane and secretory protein synthesis
Folds proteins in lumen
Glycosylates proteins
Makes disulphide bridges
Oligomerisation
Checks quality of proteins
Calcium store
Smooth er synthesises lipids

Question 16

Golgi apparatus

Answer 16

Modification of secretory and transmembrane proteins after the ER

completes glycosylation and sorting of proteins so they are packaged in to the right vesicles and sent to the right place

E.g. sent to plasma membrane or endosomes

Discovered by Camillo Golgi

stacks of membrane compartments, flattened sacs with separate internal lumen compartments that each have different enzymes inside as you progress through the stack.

Golgi is orientated all have a cis and trans face:

Incoming cis-face towards nucleus proteins to be edited

Outgoing trans-face towards plasma membrane contains mature proteins

Golgi orientation

Question 17

Golgi structure

Answer 17

Flat sac-like cisternae

Each is a distinct compartment

Cis face near nucleus incoming site

Trans face near plasma membrane outgoing site

Secretory proteins move in cis to trans direction

Usually near centrosomes in animal cells

Question 18

Golgi as part of the secretory pathway

Answer 18

Proteins synthesised in ER
Packaged into vesicles
Transported to Golgi
Glycosylated in different stacks
Packaged into vesicles
Transported to plasma membrane

ER vesicle of glycosylated protein fuses to cis Golgi network
Cis Golgi trim oligosaccharides
Medial Golgi attach additions and do further trimming
Trans Golgi add complexity to disaccharides

• enzymes mediate each stage

Retrograde transport Golgi>Golgi and Golgi> ER for retrieval of resident proteins

Question 19

Vesicle transport between organelles driven by diff vesicle forming “coat” proteins

Answer 19

COPl: Golgi to Golgi and Golgi to ER

COP ll: ER to golgi

Catherine: Golgi to endosome and plasma membrane to endosome

Question 20

COP ll vesicle formation

Answer 20

Cargo receptors recruit cargo
Sar1 (small gtpase) recruits adaptors to receptor
Adaptors (sec 23/24) bind receptors
Coat proteins (Sec13/31) bend membrane

Question 21

Golgi summary cisterna progression model

Answer 21

Glycosylates proteins
Series of separate compartments
Recieves new protein from ER in COPll vesicles at cis face

Forms cis network/cisterna by fusion with COP l coated vesicles from cis cisterna

Cis cisterna matures into medial cisterna by fusion with vesicles from medial cisterna

Medial cisterna matures into trans cisterna by fusion with vesicles from trans cisterna

This is the cisternal progression model