Lecture 8 - The secretory pathway

Question 1

How did George Palade discover the secretory pathway?

Answer 1

Through a pulse-chase experiment
-Injected [3^H]-leucine into guineapig pancreatic cells (endochrine secretory cells in the small intestine]
-then ‘chase’, removed radioactively labelled leucine and added non-radioactively labelled amino acid to chase the radioactivity through the system
-visualised where transported throughout the cell
Identified:
path of proteins - ER-Golgi-plasma membrane
proteins are always associated with membranes

Question 2

What is theprimary role of the secretory pathway?

Answer 2

protein sorting, supplies the EC space with proteins

Question 3

What are the fates of cytosolic proteins?

Answer 3

1. Remain in the cytosol

2. Reviece a targeting sequence and be directed to a specific place inside the cell or extracellular

Question 4

What occurs when a ribosome translates an mRNA containing an ER signal sequence?

Answer 4

1. partially translated protein, mRNA, ribosome are targeted to the ER where it can either become: luminal protein or polytopic/membrane protein, or go through the secretory pathway to the golgi and from there either into: plasma membrane, lysosome, or extracellular space

Question 5

What are the three fundamental ways a protein can move from one vesicle to another?

Answer 5

• gated protein transport e.g. into/out of nucleus
• membrane transport, when from one environment to a different environment
• vesicular transport, move from one environment to the same environment

Question 6

Where are proteins initially found in the Golgi that come onto be be proteins in the membrane and lumen of lysosomes/endosomes etc?

Answer 6

Membrane: proteins in the membrane of the Golgi (leading to most proteins having glycosylated areas on outside of cell)
Lumen: proteins in the lumen of the Golgi (glycosylated)

Question 7

What are features of proein sorting targeting sequences?

Answer 7

• amino acid sequences 15-60 amino acids in length
• either linear epitope or regions of amino acid sequences that form a particular signal patch when the protein folds into a 3D shape

Question 8

What are specific features of nuclear targeting sequences?

Answer 8

• often have a lot of charge (Lys and Arg residues)

- allow proteins to be taken into the nucleus (if it is a cytosolic protein)

Question 9

What are specific features of ER targeting sequences?

Answer 9

• often have basic residues (Arg, Lys, His)

- large hydrophobic patch 10-12 amino acids long

Question 10

What are specific features of mitochondria targeting sequences?

Answer 10

lysine interspaced with arginine

Question 11

What was Blobel’s signal hypothesis and experimental evidence?

Answer 11

in vito translation of RNA with ribosomes in the presence of microsomes leads to the protein being smaller
-of the proteins that had been transcribed, they contain basic amino acids followed by a stretch of hydrophobic amino acids and a signal peptidase cleavage site

hypothesis: mRNA translated into the ER and as it is the N-terminal end is cleaved off

Question 12

What is the process of entering the ER for an mRNA with a targeting signal sequence?

Answer 12

1. Ribosome binds to the mRNA and initiates polypeptide synthesis. If the protein needs to be targeted the first translated protein stretch is the signal sequence
2. The signal sequence is recognised by SRP and this binds to peptide sequence, pausing translation, and in the active form it is recruited to the ER by binding to the SRP receptor
3. Once the extended poly peptide and ribosome have been brought to the ER the SRP and SRP receptor dissociate from the ER and polypeptide.
4. Polypeptide is then extruded through ER through the Translocon and amino acids continue to be added onto the C-terminus
5. Protein is therefore targeted to the ER lumen

Question 13

What are the types of entry for a peptide to be targeted and inserted into the ER?

Answer 13

Co translational entry
- dominant
Post translational entry
-prominant in yeast
-still requires SRP, SRP receptor and translocon
-also requires sec62, 63, 71, 72 to push peptide through the translocon

Question 14

What is the structure of the Signal Recognition Peptide (SRP)?

Answer 14

-made up of 6 proteins and an RNA molecule

Question 15

What is the structure of the translocoon?

Answer 15

-heteromeric protein
-core formed by 3 Sec61 subunits:
α, β and gamma which form a channel
-3 subunits binds to the ribosome (shown through crosslinking experiments)

Question 16

What is the process of entry to the ER for transmembrane proteins?

Answer 16

1. Begins the same as the luminal proteins but amino acid sequence has areas of high hydrophobicity (matches that of the lipids in the membrane) which act as a stop translation sequence
2. Translocon places this area into the membrane and translation continues on the rest of the sequence

Question 17

What are the types of transmembrane proteins?

Answer 17

Type I e.g. LDL receptor
Type II e.g. transferrrin receptor, influenza HN protein
Type III e.g. cytochrome p450
Type IV e.g. G-protein coupled receptor, β adrenergic receptor

Question 18

What are the features of type I transmembrane protein?

Answer 18

Type I
-have N terminal seignal sequence, single transmembrane stretch of hydrophobic residues, N-terminal side of the polypeptide on the exterior of the cell membrane, C-terminal side exposed on the cytoplasmic side

Question 19

What are the features of type II transmembrane protein?

Answer 19

Type II

• have a single transmembrane stretch of hydrophobic residues with the C-terminal side of the polypeptide on the exterior of the cell membrane and the N-terminal side exposed on the cytoplasmic side
• (+) charges on the N terminal end mean that it does not easily go through the translocon and so is preferred on the cytoplasmic side
• no signal sequence

Question 20

What are the features of type III transmembrane protein?

Answer 20

Type III

• have a single transmembrane stretch of hydrophobic residues with the C-terminal side of the polypeptide on the cytoplasmic side and the N-terminal side on the luminal side
• (+) charges on the C terminal end mean that it does not easily go through the translocon and so is preferred on the cytoplasmic side
• no signal sequence

Question 21

What are the features of type IV transmembrane protein?

Answer 21

• transmembrane proteins that span the membrane multiple times
• stop sequence can be found in the region of high hydrophobicity which stops the peptide going through the membrane and allows it to move to the other side

Question 22

How can you show the location of transmembrane proteins?

Answer 22

use hydrophobicity plot

Question 23

What is the postitive inside rule of transmembrane proteins?

Answer 23

-cytosolic ends of TM domains are more (+) charged than the luminal extracellular end

Question 24

Why are chaperones necessary for protein modification in the ER?

Answer 24

chaperones, help the proten to fold, faster than in vitro

-essential for normal cellular function

Question 25

Where does protein modification in the ER occur?

Answer 25

In the lumen

Question 26

Why can protein modification occur in the lumen and not the cytosol?

Answer 26

Lumen = oxidising
Cytosol = reducing
Disulphide bond formation is necessary for the correct folding process, cannot happen in the cytosol because of free S-H groups

Question 27

What protein in necessary to correct incorrect S-S bonds?

Answer 27

Protein disulphide isomerase (PDI)

Exchanges its S-S bond for reduced substrate protein exposed S-H group from cysteine residues

Question 28

What is the importance of proline isomerisation in the correct folding of proteins in the ER lumen?

Answer 28

-most peptide bonds are in trans- configuration
but the peptide bond preceding a proline residue can adopt either a trans- or cis- configuration = interconversion
can be accelerated by PPIase enzymes (less of an energy barrier to move from the trans- to cis- configuration)
-consequently most proteins in the ER lumen have a cis- peptide bond before a proline residue

Question 29

What proteins are necessary for protein modification in the ER?

Answer 29

chaperones
protein disulphide isomerase
PPiase enzymes

Question 30

What types of glycoslyation and quality control in the ER?

Answer 30

• N-glycosylation , Asn (N) added
• Dolichol - the addition of 14 sugars onto exposed Asn residues of the growing polypeptide chain in the lumen
• N-acetly-glucosamine, mannose, glucose = glycan

Question 31

What regulation is the regulation of proper folding in the ER lumen?

Answer 31

• Glycosylation in the lumen is followed by glucose trimming events by glucosideases
• only if all of the glucose is removed can the protein exit the ER
• if not correctly folded then bind to glucosyl transferase which adds glucose back on
• glucose is recognised by calrheticulin and calnexin which hold the protein in place preventing it from exiting the ER

Question 32

What is the unfolded protein response and ER stress?

Answer 32

~80% or proteins may not fold correctly in ER (dependent on the protein)

• UPR tiggered by ER chaperones when unfolded proteins accumulate in the ER
• leads to upregulation of chaperone expression, increased export from ER and proliferation of ER (prolonged UPR leads to cells death)
• incorrectly folded proteins are then taken by chaperones to Sec61 complex (ER protein translocator) and removed from ER
• protein then ubiquitinated and retrotranslocated to the proteosome for degradation

Question 33

Unfunctional unfolded proteins responses causes what diseases?

Answer 33

cystic fibrosis

neurodegeneration

Question 34

What is the experimental evidence for the proteins that are involved in the secretory pathway?

Answer 34

Randy Shepman through Yeast random mutagenesis

• created temperature sensitive mutants
• able to identify which genes were involved in secretory pathway process
• identified secretory defect (yeast larger/heavier), vaculolar sorting defect (unable to degrade in vacuole), retograde defect (ER proteins lost no quality control)
• then did supressor screen, overexpress all the individual yeast genes in a mutant phenotype to negate the mutation

Genes identified:
Sec - secretion mutants, defects in protein secretion
Vps - vaculolar protein sorting, cannot deliver proteins to the endosomal/lysosomal system
Ret - retrograde transport, deficient in transport from Golgi to the ER

Question 35

What are the benefits of transport between organelles via vesicles?

Answer 35

• allow complete separation of proteins from cytosol

- allow transport of membrane proteins and lipids

Question 36

What is the process by which proteins exit the ER?

Answer 36

• luminal proteins bind a receptor in the ER membrane which then binds to Sec12
• Sec12 recruits and binds Sar1 (GTPase) to exchange its GDP for GTP
• in its active state Sar1 binds to the ER membrane and recruits the COPII coat proteins (Sec23, 24 heterodimer) which bind to cargo
• These recruit Sec 13 and 31 which curve the membrane, to the Sar1-GTP,Sec23/24 complex
• a ‘cage’ forms around the proteins and vesicles bud off from the ER

Question 37

What are three types of protein coats and where are they found?

Answer 37

CopII - Vesicles going from ER to the Golgi
CopI - Golgi to the ER (retrograde)
AP2 coat and clatherin - endocytosis

Question 38

How are ER resident proteins returned to the ER?

Answer 38

Through retrograde transport

• COPI coat binds to the KDEL receptor (ER soluble protein retreival signal) [lysine, aspartate, glutamate, leucine]
• ER transmembrane proteins have double lysine/arginine motif which enables proteins to be retreived by the COPI mechanisms

Question 39

What are the cisterni sections of the Golgi?

Answer 39

1. Cis golgi
2. Medial golgi
3. Trans golgi

Question 40

What are the functions of the 3 cisterni sections of the golgi?

Answer 40

High mannose glycan is transferred to Golgi form ER

1. Cis golgi - trimming mannoses
2. Medial golgi - adding GIcNAc to initiate branching
3. Trans golgi - adding galactose to NeuAc to increase complexity

Question 41

What other types of glycosylation are there in the Golgi?

Answer 41

• O-glyxosylation: initiated by the addition of GalNAc to the Ser/Thr side chains
• Proteoglycan synthesis - large O linked sulfated sugar polymers on Ser/Thr
• Glycolipid biosynthesis

Question 42

What are the two model for Golgi modification?

Answer 42

Vesicular transport model
-Enzymes contantly in the Golgi and cisterni and proteins traffic through, bud off each cisterni and passed to next
Cisterni maturation model: maturation through the golgi, enzymes bud off going through retrograde transport and get reclyed back to Cis golgi

Question 43

What are some of the funtional roles of glycans?

Answer 43

• folding in the ER
• determining blood group
• egg/sperm interactions
• immune response
• signalling
• stability/protease resistance of proteins
• molecular interaction recognition
• architechure of ECM and C-M attachment (higher motility of cells in presence of branching enzyme GlcNAcTV (easier cancer metastasis)

Question 44

What structure is responsible for sorting beyond the Golgi?

Answer 44

Trans-golgi-network

Question 45

What are the three fates of proteins post golgi sorting/TGN?

Answer 45

1. No signal, secreted from the plasma membrane through the constituative secretory pathway
2. tagged with mannose 6-phosphate and directed to the lysosome
3. can be directed to specialised secretory vesicles e.g. neurotransmitter vesicles