Cours 11 - Protein sorting

Question 1

What are the 3 types of transport that allow protein sorting?Describe

Answer 1

1. Gated transport: Cytosol to nucleus (bidirectional)
2. Transmembrane transport: mitochondrion and ER (unidirectional)
3. Vesicular transport: secretory pathway

• from ER to golgie : bidirectional
• From golgie cell/ to secretory vesicule to cell : unidirectional
• From cell surface to early endosome: bidirectional
• From early endosome to late endosome : unidirectional
• From late endosome to lysosome : unidirectional
• From late endosome to golgie: bidirectional

Question 2

What are signal sequences and patch?

Answer 2

Amino acid sequence that directs protein to specific destination.

• Signal sequence: signal is composed of consecutive amino acid sequence at terminal end of protein.
• Signal patch: signal amino acids are internal to protein and their can be multiple that, when the protein if folded, join together, forming the signal.

Question 3

Give an exemple of bidirectional transport occuring in and out of the nucleus

Answer 3

GRP synthesised in the cytosol and transported to the nucleus

mRNA synthesised in the nucleus and exported in the cytosol

Question 4

Describe the nuclear pore complex structure

Answer 4

• Perforate nuclear envelope.
• Octagonal structure.
• Composed of nucleoporins:
• ring: hold the nucleopore in the membranne,
• scaffold: forms the structure of the channel
• channel: prevent everything from diffusing, allow selective transport somehow…#@$%!*!

•aqueous pore.

as large as vesicules (to allow fully folded proteins to pass through)

Question 5

How do protein pass through the nuclear pore complex?

Answer 5

• Only watersoluable protein can pass through
• Small proteins traverse by passive diffusion
• Large proteins traverse by active transport mechanism ( tangled meshwork lines the pore to block passive diffusion of large molecules)
• Proteins are imported/exported in folded conformation.

Question 6

what do Nuclear Import Receptors do

Answer 6

• Bind to nucleoporins and nuclear localization signals (NLSs) (are specific for receptors).
• Sometimes adaptor proteins involved.
• Export works the same way, but in reverse, and utilize export receptors.

Question 7

What process allows nuclear transport in the appropriate direction? give the mecanism

Answer 7

• Import and export consumes energy. Ran (a GTPase) acts as a molecular switch.
• Ran-­GDP is in the cytosol, while Ran-­GTP in the nucleus. This gradient allows transport in the appropriate direction.

*see picture for mecanism

Question 8

How is the level of Ran-­GDP and Ran-­GTP maintain in their respective compartment?

Answer 8

• GTPase-­activating protein (GAP) hydrolyzes GTP, converting Ran-­ GTP to Ran-­GDP.
• Guanine exchange factor (GEF) promotes GDP-­GTP exchange, converting Ran-­GDP to Ran-­GTP.

Question 9

How are protein sorted (sent) to the mitochondria (don’t describe the entry process)

Answer 9

• Proteins must be encoded in the nucleus and imported from the cytosol.
• Posttranslational translocation :after the synthesis in the ribosome, it is translocated into the mitochondira
• Proteins are transported to matrix or inserted into membrane.
• Mitochondrial signal sequence is modified as an aa helix and are charged
• (+) charged amino acids are exposed at one side, uncharged ones at the other (allows recognition)

Question 10

Why would the mitochondria need proteins for?

Answer 10

•Many mitochondrial proteins are necessary for electron transport, oxidative phosphorylation, and ATP synthesis.

Question 11

Describe Protein Translocator Complexes and their role

Answer 11

1. TOM: translocase of the outer mitochondrial membrane.
   * imports all mito-­destined proteins to intermembrane space;; membrane insertion at outer membrane.
2. TIM: translocase of the inner mitochondrial membrane.
   * imports proteins to matrix (TIM 23);; insertion into inner membrane (TIM 23);; insertion of carrier proteins (TIM 22).
3. OXA complex
   * same as TIM

Question 12

How are protein inserted in the mitochondria,

Answer 12

1. Interacting proteins (e.g. chaperones, cytosolic Hsp70) maintain unfolded structure
2. Binding and recognition of signal sequence at TOM receptor proteins (TOM 20, TOM 5)
3. Transfer of signal sequence and polypeptide to TOM channel (TOM 40)
4. Interacting proteins (e.g. Hsp70) stripped away by ATP hydrolysis
5. Unfolded polypeptide fed through TOM channel
6. Polypeptide bound to TIM complex
7. Electrochemical H+ gradient in theinter membrane pulls (+) charged signal sequence through TIM (bc inside mito is -)
8. Signal sequence cleaved away by matrix processing peptidase (MPP), and mitochondrial Hsp70 binds
9. ATP hydrolysis induces conformational change of mitochondrial Hsp70, polypeptide is pulled through

Question 13

what is the Cross-­Bridge Ratchet Model

Answer 13

instead of ATP hydrolysis induce conformational change of mitochondrial Hsp70, polypeptide is pulled through one shot, hsp was going down and dissociating and another one would bind like mini ratchet slowly pulling it through,

Question 14

How are protein inserted into the Inner Membrane

Answer 14

see pic

signal charge dependent (orientation)

2 methodes, same results

Question 15

what helps to line up the TOM and TIM complexes?

Answer 15

the TIM arm that reaches the outer membrane (alpha helices)