Protein secretion and sorting

Question 1

What are organelles and how do they originate?

Answer 1

functionally distinct, membrane-bounded compartments.

First only DNA and membrane bound organelles like in PROKARYOTIC CELLS. DNA surrounded by nucleus gaining a membrane

Mitochondria initially an external prokaryotic cell that incorporates eukaryotic cell membrane.

Question 2

Name three transport mechanisms for proteins

Answer 2

1)Gated transport

occurs between cytosol and nucleus

2)Transmembrane transport

occurs where organelles distinct, ie from cytosol to ER

3)Vesicular transport

occurs between systems like ER to golgi to vesicles ect.

Question 3

How are proteins sorted/sent to different regions in the cell?

Answer 3

Proteins have SIGNAL sequences or signal PATCHES where there separate regions on the unfolded peptide that interact (become the patch) when folded.

Question 4

Describe difference between signal peptides and patches AFTER sorting.

Answer 4

Signal peptides: 15-60 aa long, often removed by specialized signal peptidases .

Signal patches remain part of the protein with a selfgovering
Function.

Question 5

What role do Nuclear Pores (NPC) have?

Answer 5

Proteins enter nucleus through nuclear pores.

Small proteins (< 5 kDa) diffuse through the pores.

Large proteins (> 5 kDa) require targeting sequences and an active mechanism

Question 6

What is the NPC and what is its structure?

Answer 6

It is a large protein complex (octagonal) containing over 30 different protein nucleoporins forming a channel for protein import and export

There is about 3000in one cell.

Question 7

Describe how Proteins are transported through the nuclear pore?

Answer 7

protein contains a NLS (Nuclear Localisation Sequence) often found interal in its structure. ie, this does not get removed.

NLS recogised by an importin receptor known as KARYOPHERIN (there are specific importins)

Exportin receptors mediate protein and mRNA out of nucleus.

Question 8

What is Ran in terms of the nuclear pore?

What is GAP and GEF and what does it do here?

Answer 8

Ran is a GTPase - a small GTP-binding protein with GTP hydrolysis activity.

Its gradient is important to active transport across the nuclear membrane; energy is produced by the hydrolysis of GTP

Its concentration is HIGH in the NUCLEUS

LOW in the CYTOPLASM.

GAP (GTPase activating enzyme) and GEF (Guanine exchange factor)

They trigger the Conversion between the stages RanGTP and RanGDP

Question 9

What machinery is used to transport proteins into the mitochondria?

Answer 9

By a twin-pore translocator

Membrane has two membranes and two compartments: intermembrane and the matrix spaces.

TOM receptor on mitochondrial outer membrane
(~9 membrane proteins in a complex)
recognises signal sequence and acts to translocate protein to the inter-membrane space.

TIM complexes (on the inner mitochondrial membrane)
TIM17-23 complex translocates proteins to the matrix

TIM22-54 complex translocates proteins to the inner membrane

Proteins passed DIRECTLY from TOM to TIM

after import Protein signal at the N (18-25) terminal is rapidly cleaved by the matrix processing protease (signal peptidase)

Question 10

What is a Amphipathic alpha-helical helix?

Answer 10

cluster of positively helix where there are charged aa

on one side and hydrophobic cluster on the opposite side;

Question 11

Describe the energy gradients involved in protein mitochondria transport.

Answer 11

1) First,in the cytoplasm, Hsp70chaperones on the importing protein are taken off by ATP hydrolysis (ATP made).
2) Next an Electrochemical H+ gradient is used to drive translocation through the TIM
3) Finally, Hsp chaperones IN THE MT are taken off the imported protein by hydrolysis of ATP (making ADP)

Question 12

What determines which proteins end up where in the Mt?

Answer 12

Normally, proteins taken to matrix.

but Proteins residing in the inter-membrane space or the inner membrane such as CYTOCHROME C1, require multipart signals with additional information

CYTOCHROME C1 has 61 amino acid leader:
first 32 specify matrix localisation when fused to other proteins (uncharged/basic)

The next 19 amino acids target protein to remain in its proper location inner membrane (uncharged/hydrophobic)

Question 13

Describe the process of protein translocation secretion involving the ER.

Answer 13

This involves Budding and fusion of membrane-bound vesicles.

Translocation across the ER membrane is through a GATED, protein-lined pore or translocon which is mediated by a SRP (SIGNAL RECOGNITION PARTICLE)

The N terminal leader sequence o the protein is made of HYDROPHOBIC residues.

After the proteins translocation through the membrane, the signal is cleaved.

Question 14

Describe the E.R.

Answer 14

It is a netlike web of branched tubules

Its has an important role in lipid and protein formation; almost all proteins are processed through the E.R

There are ribosomes on Rough E.R surface.

Question 15

Step by step of the process of protein secretion involving the ER.

Answer 15

1) Ribosome starts protein synthesis
2) SRP attaches to new proteins hydrophobic, n terminal leader (12-20) sequence and stops translation at about 70 aa.
3) SRP binds to its receptor, then ribosome attaches to the E.R membrane and translation starts again.
4) Leader sequence enters the membrane and the protein follows.
5) Leader sequence is cleaved
6) translation continued until entire protein is secreted.
7) ribosomes released from mRNA.

Question 16

What is the SRP in E.R secretion?

Answer 16

Signal Recognition Particle is a ribonucleoprotein complex of 7S RNA + 6 proteins that binds to the proteins signal sequence and then to a SRP receptor on the E.R membrane.

Its conformation changes when it binds to the signal as it not only binds to the signal (at SRP54) but bends to also bind to the ribosome (SRP19-ELGONGATION FACTOR) the protein is emerging from.

Question 17

What is the Translocon in E.R secretion?

Answer 17

Translocon is a pore in the membrane of the E.R containing a channel.

Consists of Sec61 core (alpha, beta and gamma subunits) with gates on both sides.

There is a gate on both sides.

Ribosome seals from cytoplasm side with SRP.

Internal E.R side opens when protein synthesis is resumed.

Protein goes through channel from cytoplasm into lumen of E.R

Question 18

List types of membrane proteins

Answer 18

1) Peripheral- not embedded but have ionic interactions with protein/lipid head groups.
2) Integral-inserted into membrane and can be transmembrane.
3) Lipid and GPI anchored proteins

The TMD of a new protein gains access to the lipid bilayer through a TRANSLOCON channel formed by the helixes of the TMD

Question 19

What is a Transmembrane domain (TMD)?

Answer 19

TMD adopts an α-helical conformation

A helix of 21-26 hydrophobic/non-polar amino acids that can ‘DISSOLVE’ in the lipid membrane.

R groups (usually hydrophobic aa) face out into the lipid while the Hydrogen bonded AMIDE bonds face inside the helix.

Different TMDs may prefer different surrounding lipids.

Question 20

Describe two types of transmembrane protein.

Answer 20

Type 1: (C-terminus on cytosolic side) signal peptide cleaved but transfer stops at a second hydrophobic N domain (“stop transfer” sequence) that becomes the TMD.

Type II: (N-terminus on the cytosolic side) signal peptide remains membrane-associated but orientation of polypeptide is flipped during the translocation process (“signal anchor”)

The more POSITIVE end of the TMD is on the INSIDE CYTOPLASM whether it is type 1 or 2 or not.

Question 21

What is a MULTI transmembrane protein

Answer 21

Proteins with multiple TMD, the number of which will determine the orientation of the protein.

If there is an EVEN number of TMD, both N and C ends will be on the same side.

UNEVEN number of TMD will lead to N end being on the opposite side from C.

Question 22

Give an example of a single TM protein.

Answer 22

Glycophorin:131 AA long.

It consists of an ectodomain (with the N terminal), transmembrane domain (23 aa long) and a cytoplasmic domain (with the C terminal)

I is a TYPE 1 TMD protein as the N termal is OUTSIDE.

It is very GLYCOSYLATED

Question 23

What is a Multi-pass TM protein

Answer 23

One protein structures span the bilayer several times (several strands) where there be POLAR and charged aa.

TMD may be doughnut shape forming a pore with these POLAR aa facing eachother; channels formed that can transport polar molecules (glucose)

Question 24

Give an example of a multipass protein.

Answer 24

Bacteriorhodopsin; consists of 7TM alpha helices.

Type 1: as N terminus OUTSIDE membrane.

Question 25

In what ways is a multipass protein amphipathetic? Give an example.

Answer 25

The pitch of the α-helix of a TM protein allows hydrophobic and hydrophilic faces to form making it into an amphipathic helix.

Example: GLUT 1 with 12 helices in the membrane (Type 2)

5 HELICES form a central PORE that is HYDROPHILIC, with hydrophobic residues on the outside. 9charge residue interact with eachother.

Question 26

What is a GPI anchor?

Answer 26

glycosyl-phosphatidylinositol (GPI) anchor is a GLYCOLIPID on the outside of cells that binds to protein via the GPI transamidase (in the membrane).

Proteins with anchors can DIFFUSE WITHIN MEMBRANE.

Question 27

Give types of lipid side chain aditions that help anchor proteins

Answer 27

N-myristoylation
addition of C14 saturated fatty acid to an N-terminal glycine
irreversible

Palmitoylation
addition of a C16 saturated fatty acid to a cysteine
reversible membrane association

Question 28

Describe the role signals have in their pathway for secretion.

Answer 28

Packaged into vesicles to Golgi

After Golgi vesicles carry proteins to cell membrane or a lysosome.

To be directed to the lysosome proteins need a Mannose-6-phosphate (phosphorylated sugar) modification

SNARES are parts of the protein that tell vesicles where to go

To remain in the E.R, proteins must have EXTRA signals like a KDEL signal at the C end.

Question 29

Name different types of coated vesicles AND what are they controlled by?

Answer 29

Coat proteins assemble a basketlike lattice molding the forming vesicle
1) Clathrin (fused with plasma membrane)

2) COPI-coated (on golgi and early endosome
3) COPII-coated (on E.R)

Monomeric GTPases control coat assembly/recruitment: determines vesicle transport (with GAP and GEFs.) and

Question 30

In what way do SNARE proteins and Rab GTPases guide membrane transport?

Answer 30

Vesicles are very selective and only fuse with the right membrane; this is controlled by SNAREs and GTPases called Rab; membrane and vesicle markers are COMPLEMENTARY where v-SNAREs and target membrane SNARES combine.

Specificity is ensured by RAB proteins

some neurotoxins (tetanus, botulism) are proteases cleaving SNARE proteins and block synaptic transmission

SNARES are RECYCLED through their disassembly with NSF.

Question 31

What is Proteolysis?

Answer 31

IRREVERSIBLE PROCESS of Destruction of proteins:
1) taken up by endocytosis

2) misfolded proteins
3) during the cell cycle and Apoptosis (Regulated)
4) during the presentation of antigens by the immune system
5) processing

Question 32

Describe Proteolytic Processing of insulin

Answer 32

Signal peptide of the initial peptide are removed to form proinsulin.

Disulphide bonds form between A and C chain.

B chain is removed

Question 33

Give some details on the lysosomes in proteolysis

Answer 33

They contain acid hyrolases such as proteases and lipases.

They use ATP hydrolysis to gain protons through a proton pump.

Question 34

What is a proteasome?

Answer 34

It is a Cylinder consisting of 4 rings (2 beta inner, 2 alpha outer) each of 7 identical subunits.

There are 3 central cavities with gated entry where proteins are broken down.

N-terminal helix of each α subunit restricts entry.

Question 35

In the proteasome, which subunits are responsible for the breakdown of proteins?

Answer 35

The beta subunits. B1, 2 AND 5.

B5 MOST IMPORTANT (B1 least)

β1 : acidic (peptidyl-glutamyl-peptide splitting) activity
β2 : trypsin-like (cleaving after basic residues)
β5 : chymotrypsin-like (cleaving after hydrophobic residues)

Products are 4 to 14 aa.

Question 36

Why would a Immunoproteasome be used?

Answer 36

For antigen processing for MHC I presentation at cell surface (viral infection response) where beta subunits are replaced by isoforms.

Question 37

What is the two REGULATORY CAP on either side of a proteasome needed for?

Answer 37

Full proteasome is 26S - 20S core plus two 19S regulatory Cap structures that are needed to recognise proteasome substrates usually marked with poly-ubiquitin chains
(added destruction flags)

Ubiquitin is removed and recycled (DUB activity) and protein enters 20S core for degradation.

Ring/base of cap involved in gated entry of selected substrate into the proteasome core.

Roles of 19S Cap in UNFOLDING and transmission of substrates

Question 38

What is Ubiquitin?

Answer 38

76 amino acid polypeptide on which the LYSINES other peptides (needing to be destroyed) can bind to and form poly-ubiquitin chains.

Peptides bind to LYSINE48 of ubiquitin.

Question 39

Describe Ubiquitylation

Answer 39

Ubiquitylation involves E1(ubiquitin-activating enzyme), E2 (Ubiquitin-conjugating enzyme) and E3 (ligase) enzymes

ubiquitin binds to target lysine by ISOPEPTIDE bonds. This is reversible.