7: Protein Sorting and Intracellular Traffic

Question 1

endoplasmic reticulum (ER)

Answer 1

rough ER - has ribosomes
smooth ER - no ribosomes
nuclear envelope continuous with ER

Question 2

SER

Answer 2

synthesis of lipids
synthesis of steroid hormones
storage and release of Ca2+ from sarcoplasmic reticulum (specialised version of ER)
detoxification - lots of SER in liver for alcohol

Question 3

RER

Answer 3

site of protein synthesis and processing for transmembrane proteins which are destined for the ER, Golgi, plasma membrane lysosomes or endosomes
also site of protein synthesis and processing for proteins secreted from the cell
other ribosomes are located in the cytosol which synthesis other proteins

Question 4

protein synthesis on ER

Answer 4

pool of large and small subunits come together to form ribosome
mRNA threads through ribosomes and protein emerges
ribosome is bound to RER so protein goes straight into ER

Question 5

co-translational translocation

Answer 5

is when membrane-bound ribosomes insert growing polypeptide chains directly into an ER
protein remains in primary sequence because one end is attached to ribosome
for co-translational translocation, the signal sequence must by recognised (SRP), and be recognised by the ER and embedded within the ER membrane (SRP receptor and translocator)

Question 6

SRP = signal recognition particle

Answer 6

SRP recognised the signal sequence on the N-terminal of the growing polypeptide
it binds to the signal sequence and the ribosome temporarily pausing synthesis

Question 7

ER recognition

Answer 7

SRP receptor sits on RER membrane and binds with SRP to tether entire structure to the membrane
protein begins to move through lumen on protein translocator
SRP releases itself, ribosome left separate with protein on membrane
protein feeds through translocator unit it enters ER and folds
ribosomes can tether and untether themselves from RWE to get proteins
mRNA from ribosome may remain bound to ER until translated, then degraded

Question 8

exocytosis

Answer 8

if a protein is released from a cell (exocytosis) then it will be fully transported into ER lumen
if a protein is destined to be a membrane protein, it will be embedded within ER membrane

Question 9

once the protein is inside the ER

Answer 9

it will fold into its 3D confirmation via: ionic bonds, hydrogen bonds, van de waals attraction, disulphide bond formation
if the protein cant fold up it goes through glycosylation

Question 10

glycosylation

Answer 10

refers to the addition of a sugar to a protein
three functions: quality control, recognition, protection
a precursor oligosaccharide composed of 14 sugars is added to the protein via the N-terminus

Question 11

quality control

Answer 11

3x glucose and 1x mannose cleaved from the oligosaccharide
if it doesn’t fold correctly, glucosyl transferase enzymes adda a single glucose unit back to chain
calnexin (a chaperone protein) binds to unfolded protein to prevent aggregation
removal of the terminal glucose (glucosidase) releases protein from calnexin
glucosyl transferase determines if protein is correctly folded, if not a single glucose is added back to re-enter cycle

Question 12

unfolded protein response = when protein remains misfolded

Answer 12

misfolded protein causes ER stress and triggers unfolded protein response (UPR)
1. inhibits protein synthesis
2. degrades misfolded protein
3. increases transcription of chaperone proteins
if problem persists apoptosis occurs which is programmed cell death

Question 13

George Emil Palade

Answer 13

won 1974 Nobel prize for defining the cell secretory pathway
pulse chase experiments:
- addition of radiolabelled amino acids to cell
- all newly formed proteins are radiolabelled (pulse)
- the cells then return to non-radioactive medium (chase)
- after 7 mins = newly formed proteins no longer in RER, in Golgi
- after 2 hours = in vesicles seen leaving Golgi

Question 14

3 intracellular membrane traffic pathways

Answer 14

secretory pathway = how cellular cargo can be moved from ER (can go through Golgi then leave or go into endosome)
endocytic pathway = things coming back into the cell
retrieval pathway = useful components that can be recycled

Question 15

type of vesicles

Answer 15

COPI coated vesicles = released from Golgi
COPII coated vesicles = released from the ER
clathrin coated vesicles = from the plasma membrane and between Golgi and endosomes

Question 16

ensuring specificity

Answer 16

how to proteins end up in the right place?
GEF found on vesicle membranes activates Rab-GDP in the cytosol to form Rab-GTP
Rab-GTP binds to Rab effector protein on the target membrane, tethering off the vesicle to the target membrane
lots of Rabs and effector proteins ensure specificity

Question 17

membrane fusion

Answer 17

vesicle membranes and target organelle membranes must fuse using SNARE proteins
V-SNAREs (vesicle) and T-SNAREs (target) are specific to one another providing more specificity
wrap around each other to form a stable trans-SNARE complex
once within 1.5nm, phospholipids can flow between

Question 18

ER to Golgi

Answer 18

COPII coated vesicles bud off from ER carrying cargo
multiple vesicles can fuse if SNAREs match to form vesicle tubular clusters

Question 19

Golgi Apparatus

Answer 19

Golgi is a stack of flattened membrane enclosed compartments called cisternae
has a cis (retrieves things from ER) and trans (secretes this onwards) face
processes oligosaccharide chains to promotes correct folding
sorts and targets proteins for correct pathway

Question 20

Golgi to lysosomes

Answer 20

lysosomes are organelles that degrade unwanted material
contain lots of enzymes to break down macromolecules
only active at pH 4.5-5 to stop enzymes breaking down anything if lysosome splits
delivered via clathrin coated vesicles
lysosomes tagged by mannose-6 phosphate
M6P directs proteins to the lysosomal network

Question 21

endosomes

Answer 21

late endosomes contain material that has been ingested by the cell
fusion with either endolysosomes or lysosomes creates endolysosomes
once digestion of endocytosed material is complete, they form lysosomes

Question 22

endocytosis

Answer 22

after an endocytic vesicle fuses with an early endosome the injested material is either…
1. degraded = moves towards centre of cell to alter membrane composition, starts degradation
2. recycled = vesicles containing material for recycling to plasma membrane bud off and fuse with recycling endosome

Question 23

exocytosis

Answer 23

get cargo out of cell
constitutive secretory pathway = all cargo released at once
regulated secretory pathway = cargo maintained within secretory vesicles until it’s needed