Liu 1

Question 1

Does the orientation of protein and lipids in the membrane change during transport?

Answer 1

No, topological relationship maintained

Question 2

Transfer into nuclear envelop is through _____

Answer 2

NPCs (nuclear pore complexes)

Question 3

What is the size limit for NPC?

Answer 3

60 kDa for passive diffusion (gated diffusion)

-larger molecules pass by active transport

Question 4

What allows specificity of nuclear import?

Answer 4

Nuclear localization signal (NLS) - mostly lys and arg

-can transport folded protein through pore unlike normal membrane transport

Question 5

What is Ran?

Answer 5

GTPase required fro nuclear import and export

1) cytosol: Ran-GDP is imported to nucleus where Ran-GEF removes GDP
2) GTP binds forming Ran-GTP which is exported to cytosol
3) Ran-GAP hydrolyzes GTP to make Ran-GTP again

Question 6

How does Ran provide directionality to nuclear transport?

Answer 6

Import

1) protein imported
2) Ran-GTP binds after cargo delivered and allows export of receptor
3) Ran-GTP is hydrolyzed and Ran-GDP dissociates from receptor

Export (same)

1) Ran-GTP binds to receptor with cargo inside mito, which is exported
2) In cytosol, product is released and Ran dissociates as Ran-GDP

Question 7

How is protein transported into mitochondria?

Answer 7

1) Precursor with protein and signal sequence recognized by TOM (transporter outer membrane complex)
2) Fed through TIM complex (transporter inner membrane) and the signal cleaved and mature protein folded

Question 8

Peroxisome protein transport?

Answer 8

Has signal at C-terminus that isn’t cleaved

-imported without being unfolded

Question 9

co-translational vs post-translational translocation?

Answer 9

co-translational: in ER (translational + translocate together)
post-translational: mito, nuclei, peroxisome (translational then translocate)

Question 10

who were blobel and dobberstein? hypothesis?

Answer 10

showed co-translational translocation
mRNA + reticulocyte + rough microsome -> 25 Kd, protease resistant
-no microsome or microsome added after protein synthesized showed longer protein that also protease sensitive

-signal directs ribosome to translocator, as translation continues, signal peptidase clips off signal sequence, so shorter peptide

Question 11

What recognizes the ER signal? Important features?

Answer 11

SRP - signal recognition particle (recognizes N-terminal signal sequence)
signal-sequencing binding pocket: lined by methionines; pocket for hydrophobic signal sequences

translational pause domain: pauses translation when signal recognized; until it reaches ER membrane receptor

Question 12

How is single-pass protein integrated into ER membrane?

Answer 12

-has start-transfer signal sequence (hydrophilic), and stop transfer (hydrophobic). Stop transfer stops translocation there.

Question 13

What is no N-terminal signal and only internal signal?

Answer 13

Still recognized by SRP, oriented based on charged AA near sequence. (+ on cytosol, - on ER lumen side) N and C terminus can be either way

Question 14

What about double pass? multipass?

Answer 14

• two internal sequence, one start and one stop
• in multipass, just repeats start/stop sequences

REMEMBER SRP AND RECEPTOR ARE GTPASE

Question 15

What is ERAD pathway? Disease?

Answer 15

ER-associated degradation

• misfolded protein exported
• degraded by proteasome after ubiquination
• aberrant degradation can cause diseases

Cystic fibrosis and Parkinson’s disease
PD: mutation of E3 ubiquitin ligase in ERAD causes juvenil parkinson’s
CF: mutation of phenylalanine in CF conductor regulator; due to missing phenylalanine
-can work but recognized as misfolded and degraded

-so lack of protein because protein is degraded or accumulation due to defective ERAD

Question 16

Where does glycosylation occur?

Answer 16

All protein in ER and subsequent vesicles

Question 17

3 types of glycoproteins?

Answer 17

1) o-linked to serine or threonine hydroxyl
2) N-linked to amide nitrogen of asparagine
3) GPI-anchored, linked to carboxyl terminal AA of protein via a phosphorylethanolamine

Question 18

How does N-glycosylation occur? Formation of high-mannose?

Answer 18

1) transfer of oligosaccharide from dolichol-P-P-oligosaccharide to Asn on peptide (all glycoprotein share common pentasacch Man3GlcNAc2)

high-mannose

1) in ER, remove glucose
2) in golgi, high mannose has mannose removed and is processed by adding new sugars to make complex oligosaccharide

Question 19

GPI-anchored glycoprotein transport?

Answer 19

As protein enters ER lumen, GPI binds to carboxyl end of peptide (ends up on surface of membrane or in cytosol); cleavage of preexisting C terminal