Lecture 13

Question 1

List the 3 topological compartments, what major organelles/structures they are associated with, and how they communicate

Answer 1

Nucleus and Cytosol: communicate through the “gated transport” of the nuclear pore complex

Organelles in the secretory/endocytic pathways (ER, Golgi, Endosomes, and lysosomes): communicate through “budding and fusion” of vesicles

Mitochondria: not big on communication (keep to themselves bc of the potential danger from their free radicals

Question 2

In terms of protein trafficking, define Gated Transport

Answer 2

What occurs, via nuclear pore complexes that conduct active and free diffusion, between the nucleus and the cytosol

Question 3

In terms of protein trafficking, define Transmembrane Transport

Answer 3

When membrane translocators directly transport proteins from the cytosol across a organelle membrane

Question 4

In terms of protein trafficking, define Vesicular Transport

Answer 4

Membrane-enclosed transport intermediates that move proteins between various compartment via vesicles

Question 5

Sorting signals are stretches of ________ that are localized on the ______ or ______ within a protein sequence.

Answer 5

AA’s (typically 15-60 residues long) ; N terminus ; C terminus

Question 6

Define a signal patch in a protein

Answer 6

multiple, scattered sorting signals in proteins

Question 7

Signal sequences are _____ and _____ for protein targeting

Answer 7

necessary ; sufficient

Question 8

Describe signal peptidase’s role in protein sorting

Answer 8

signal peptidase may remove the sorting signal once the protein reaches it’s final destination so that it is no longer targeted

Question 9

What role do complementary receptors play in protein sorting? what is most important about a protein signal sequence?

Answer 9

they recognize signal sequences

the physical properties of the sequence are most important, not necessarily the actual sequence.

Question 10

(Signal Sequences)

describe the signal sequence that codes for the import of a protein into the nucleus.

Answer 10

Lysine and Arginine rich sequences in ANY order

the sequential positive charges from lysine and arginine is the main physical characteristic that codes for import into the nucleus

Question 11

(Signal Sequences)

describe the signal sequence that codes for the import of a protein into the Mitochondria.

Answer 11

A combination of positively charged, and hydrophobic AA’s that form a coil

ex. +H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe
(+H3N is the N terminus)
(Arg is positively charged)

Question 12

(Signal Sequences)

describe the signal sequence that codes for the import of a protein into the ER.

Answer 12

a stretch of hydrophobic AA’s

ex. Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp

Question 13

(Signal Sequences)

describe the signal sequence that codes for the return of a protein to the ER.

Answer 13

KDEL is the specific combination that is needed to signal for this ; Lysine A

ex. -Lys-Asp-Glu-Leu-COO-
(COO- is the C terminus)

Question 14

Describe the characteristics of nuclear transport (3 of them)

Answer 14

gated, bidirectional, and selective

Question 15

Proteins needed in the nucleus are imported from the _______ where they are synthesized. Alternatively, ______ and ______ molecules synthesized in the nucleus are exported to the cytosol.

Answer 15

Cytosol ; tRNA ; mRNA

Question 16

Describe Nuclear Pore Complexes (NPC’s) in terms of their composition and structure.

Answer 16

composed of 30 different nucleoporins (a type of protein) arranged in octagonal symmetry or “baskets” with one or more aqueous pores.

Question 17

List the types of transport that NPC’s conduct, and the significance of the NPC fibrils.

Answer 17

NPC’s conduct bidirectional passive diffusion of small molecules and facilitated transport of larger molecules.

fibrils extend into the cytosolic side of the nuclear membrane and bind to the particles being transported via facilitated transport. This aids in their movement across the nuclear membrane.

Question 18

Describe Nuclear localization signals (NLS’s)

Answer 18

short sequences of positively charged AA’s (lysine and arginine) that are located on many different sites on a protein and code for the selective import of that protein into the nucleus.

Question 19

What recognizes nuclear import signals (NIS’s)? describe this.

Answer 19

Nuclear import receptors (NIR’s), which are soluble cytosolic proteins that bind to NLS’s (Nuclear localization signals) and to NPC (nuclear pore complex) proteins present on their fibrils.

Question 20

NPC proteins have ________ which serve as binding sites for import receptors.

Answer 20

phenylalanine glycine (FG) repeats

Question 21

describe how receptors and their protein cargo traverse NPC’s.

Answer 21

receptors plus their cargo move through the NPC by binding, dissociating, and re-binding to adjacent FG repeats until the cargo can be released inside of the nucleus and the NIR (nuclear import receptors) then returns to the cytoplasm.

(sort of “crawls” by binding over and over to the NPC FG repeats)

Question 22

Describe what Ran-GAP and Ran-GEF are, and where they are found in the cell.

Answer 22

Ran-GAP exists in the cytosol and removes a phosphate from Ran-GTP to create Ran-GDP

Ran-GEF exists in the nucleus and adds a phosphate to Ran-GDP to create Ran-GTP

(both are Ran specific regulatory proteins)

Question 23

Describe the mechanism of nuclear import of a protein cargo and what happens to the nuclear import receptor.

Answer 23

the cargo protein bound to a nuclear import receptor enter the nucleus, and the protein cargo is released when Ran-GTP binds to the nuclear import receptor.

The Ran-GTP complex then moves out of the nucleus and is converted, by Ran-GAP, to Ran-GDP which then releases from the nuclear import protein.

Question 24

Describe the mechanism of Nuclear export of a cargo protein and what happens to the nuclear export receptor.

Answer 24

The nuclear receptor enters the nucleus through the NPC, once in the nucleus, Ran-GTP and the cargo protein bind to the nuclear export receptor.

Ran-GTP is converted to Ran-GDP and a phosphate by Ran-GAP hydrolyzing it.

the Ran-GDP and protein cargo are released in the cytoplasm

Question 25

Some shuttling proteins contain both NLS(nuclear localization signals) and NES(nuclear export signals). how are these types of shuttling proteins classified?

Answer 25

the relative rate of transport determines the classification of these proteins.

if the rate of import is greater than the rate of export, then it is considered nuclear, and vice versa.

Question 26

What happens if the rate of import/export of a shuttling protein is changed?

Answer 26

the localization (classification) of the protein will change according to the rate of import/export that is changed.

Question 27

Describe the transport of gene regulatory proteins. What are a few mechanisms that are used to accomplish this?

Answer 27

it is stringently controlled by NLS and NES signals being turned on and off.

mechanisms include Phosphorylation, proteolysis, and binding to inhibitory proteins

Question 28

describe the control of nuclear import of NF-AT during T cell activation. Include Calcineurin in this.

Answer 28

antigens cause an increase in Ca2+ in the cytosol, which dephosphorylates NF-AT and expose it’s nuclear import signal. At the same time, Calcineurin is a protein that is binding to and blocking the nuclear export signal of NF-AT.

once inside the nucleus, NF-AT activates gene transcription to activate T cells. Calcineurin leaves. when Ca2+ levels decrease.

this exposes the nuclear export signal on NF-AT and simultaneously the nuclear import signal is covered via phosphorylation.

Question 29

describe the regulation of cholesterol biosynthesis.

Answer 29

in high cholesterol cells, Cholesterol molecules bind to SCAP, which is bound to SREBP, signaling that there is sufficient cholesterol.

in low cholesterol, Golgi protease (at 2 sites) cuts the SREBP into a small released factor that goes to the nucleus and turns on cholesterol-producing genes through various biosynthesis pathways.

Question 30

What are NIR’s?

Answer 30

(nuclear import receptors) are soluble cytosolic proteins that bind to NLS on protein and to NPC proteins on fibrils that extend into the cytoplasm.