Secretory Pathway

Question 1

Theory

Answer 1

Newly synthesized proteins have a built in signal directing them to the ER and through a membrane channel to their destination
Signal peptide guides the positioning of the ribosome in the membrane

Question 2

3 secretory options

Answer 2

1. no signal sequence = released into cytosol
2. organelle specific sequence = imported into organelle
3. ER signal sequences = enter the ER/secretory pathway

Question 3

Key Experimental Evidence

Answer 3

The same piece of mRNA synthesized by free ribosomes is longer than than of bound ribosomes in the rER, telling us that the rER modifies proteins
‘signal hypothesis’ tells us that the size difference between ER and free ribosome synthesis is caused by the signal sequence being cut off by signal peptides

Question 4

Signal Sequence

Answer 4

Acts as a ‘postcode’ to direct a protein to the ER
Signal for ER translocation is N-terminal positive, hydrophobic and polar
signal recognition particle and SRP receptor aid in guiding the signal sequence to the membrane
- the hydrophobic non-polar amino acid in SS binds to the hydrophobic pocket lined with methionine in the SRP

Question 5

2 Methods of ER translocation

Answer 5

1. co-translational translocation

2. post translational translocation

Question 6

Co-translational translocation

Answer 6

1. mRNA meets ribosome and makes protein
2. signal recognising particle recognises signal sequence and stops protein synthesis
3. complex is directed to the ER
4. binds to SRP receptor/SEC translocon
5. synthesis resumes and protein is translated into the ER lumen
6. signal peptidase cleaves the signal sequence into the organelle membrane
   Energy for this process comes from the energy used in translation itself

Question 7

Post translational translocation

Answer 7

‘Ratchet’ model
BIP protein hydrolyses ATP to pull protein through the membrane - this prevents backslide
- protein deposits on the protein as it comes through the membrane
- chaperone proteins in cytosol prevent folding
Signal sequence on polypeptide associates with SEC translocon

Question 8

SEC Translocon

Answer 8

Heterotrimeric complex (a,B,y subunits)
Passive water-filled pore closed by a ‘plug’ when unused
Polypeptide fed unfolded through
Core can also open like a clamshell to let cleaved signal sequence out as well as to integrate transmembrane proteins
Hydrophobic residues that form transmembrane proteins are shunted to the side
-Sec61 core
- dynamic movement

Question 9

Golgi

Answer 9

Has intrinsic directionality (cis face receives proteins and trans face exports them)
Fused vesicular tubules with proteins/lipids enter
Proteins and lipids exit to cell surface or other organelles
Dynamic movement
Is important in protein glycosylation

Question 10

Endoplasmic Reticulum Functions

Answer 10

Lumen is an oxidative environment, so proteins can form disulfide bonds which is key for their tertiary structure

• assembly of quaternary structure
• cleavage of specific sites
• addition of GPI anchor
• cell membrane lipid synthesis
• n linked glycosacchride addition

Question 11

2 models of movement through the Golgi

Answer 11

1. Vesicular transport between cisternae

2. Cisternal maturation model

Question 12

Lysosomes

Answer 12

Simple compartment full of hydrolytic enzymes
Internal ph of 4.5-5.5 caused by ATPase pumping H ions into the interior of the organelle
The ph dependence of the enzymes protects the cell
M6P signal marks lysosome as destination for proteins
- signal for M6P unit addition to oligosacchrides is an amino acid cluster - called a signal patch
- GlcNAc phosphotransferase binds to signal patch and cleaves the GlcNAc to expose the signal
* pathogens can reduce lysosomal activity to protect themselves *
Lysosomes are viewed as a mature endosome and fuse with vesicles to destroy contents

Question 13

Single Pass Transmembrane Proteins

Answer 13

Translocation begins normally but is stopped by an additional hydrophobic segment
Lateral gate mechanism transfers this stop transfer sequence (signal sequence in the protein) into the bilayer so 1/2 of the protein is on each side of the bilayer

Question 14

Multi Pass Transmembrane Proteins

Answer 14

Determined by the combination of hydrophobic stop and start transfer signals
These sequences lock themselves in the membrane as membrane spanning alpha helices
Internal ER signal sequence acts as a start transfer sequence

Question 15

Cisternal Maturation Model

Answer 15

Golgi cisternae are dynamic structures that mature from early to late by acquiring or losing specific golgi resident proteins
The retrograde transport of golgi enzymes via COPI vesicles maintains their distribution
Experimental evidence supports this theory - shows that when cisternae are labelled with color depending on the enzymes present, they change color over time as they mature

Question 16

Alternative Views

Answer 16

Cisternae are long lived structures that retain their set of proteins and proteins are transported from one to the next via vesicles
experimental evidence shows that cargo molecules are present in small COPI coated vesicles/these vesicles can deliver them to the golgi cisternae over long distances/aggregated membrane proteins in the cisternae stay in place while soluble proteins move through

Question 17

Likely Model of Golgi Development

Answer 17

Aspects of both models are true
Stable core of long lasting cisternae in the center with edge regions undergoing maturation
matured pieces break off and fuse with downstream cisternae, transporting cargo with it
COPI vesicles transport small cargo forward and retrieve and return escaped enzymes

Question 18

Lysosomal Hydrolyase Signals

Answer 18

• contain N linked oligosacchrides covalently modified in a unique way in the cis golgi to phosphorylate their mannoses
• the M6P groups are recognised by a M6P receptor protein in the trans network that delviers them to endosomes
• low endosome ph and phosphate removal causes the hydrolases to dissociate from receptors