1.9: Protein Sorting

Question 1

what features are specific to animal cells and what do they do

Answer 1

ecm (extracellular matrix): specialized material outside of the cell
lysosome: degradation cellular component that no longer necessary

Question 2

what features are specific to plant cells and what do they do

Answer 2

chloroplasts: photosynthesis
vacuole: 2 types: storage and the other functions similarly to the animal lysosome
cell wall: tough protective outer coat

Question 3

state the 2 processes of protein sorting

Answer 3

1. post translational process
2. co translational process

Question 4

describe the post translational process of protein sorting

Answer 4

proteins fully synthesized in the CYTOSOL before sorting. the unfolded goes to mitochondria and plastids and folded foes to nucleus and peroxiomes

Question 5

describe the cotranslational process of protein sorting

Answer 5

happens in the ER, has proteins with er signal sequence (associated w er during protein synthesis)

Question 6

list the 3 sorting mechanisms of protein

Answer 6

gated transport, transmembrane transport (req prot translocators), vesicular transport

Question 7

which of the protein sorting mechanisms is restricted to one way

Answer 7

transmembrane transport

Question 8

gated transport involves protein moving between ? and which complex

Answer 8

involves proteins moving between cytosol and nucleus

involves the nuclear pore complex (NPC) which facilitates the selective transport of macromolecules and free diffusion of small molecules (<5000 daltons)

Question 9

t/f the npc can transport in both directions

Answer 9

true. nuclear import dictates from cytosol to nucleus and export is from nucleus to cytosol

Question 10

what is the npc made up of

Answer 10

nucleoporins

Question 11

describe the process of nuclear import

Answer 11

involves the nuclear import receptor which binds to NLS (rich in lys and arg) and binds to nucleoporins in npc, facilitates transport into the nucleus.

cargo proteins have the nuclear localization signal (NLS)

Question 12

the nuclear import adaptor protein has _______ <– and is the blank the same for all proteins

Answer 12

the nls, no the nls is not the same for all cargo proteins

Question 13

describe the process of nuclear export

Answer 13

involves nuclear export receptor, which is structurally related to the nuclear import receptor, binds to nuclear export signal (NES), binds to nucleoporins in the npc, transport into cytosol

cargo proteins have a NES

Question 14

which are common molecules that have a NES (3)

Answer 14

newly assembled ribosomal subunits, rna, proteins w regular nuclear import and export

Question 15

ran gtpase cycles between ____ bound and _____ bound

Answer 15

gtp and gdp bound

Question 16

what is ran gtpase regulated by?

Answer 16

• rap gap (gtpase- activating protein): stimulates gtp hydrolysis by ran
• ran gef (guanine nucleotide exchange factor): promotes exchange of gdp for gtp by ran

Question 17

what ratio is critical for direction of transport

Answer 17

[ran gtp] in nuclear; [ran gtp] in cytosol
(high; low)

Question 18

ran-gap is in the ________
ran-gef is in the _________
high [ran-gtp] in the nucleus; low [________] in the cytosol

Answer 18

ran-gap is in the cytosol
ran-gef is in the nucleus
high [ran-gtp] in the nucleus; low [ran-gtp] in the cytosol

Question 19

how does ran gtpase move between the nuclear and cytosol

Answer 19

• ran-gtp to cytosol with nuclear import/export receptors
• ran-gdp to nucleus, transported by ntf2 (nuclear transport factor 2)

Question 20

detail the steps of nuclear import of cargo proteins

Answer 20

1. nuclear import receptor binds cargo in cytosol
2. receptor + cargo move to nucleus
3. ran-gtp binding causes cargo release
4. empty import receptor + ran-gtp move to cytosol
5. ran binding protein and ran-gap promote gtp hydrolysis and release of import receptor

Question 21

detail the steps of nuclear export of cargo proteins

Answer 21

1. nuclear export receptor binds ran-gtp and cargo in nucleus
2. receptor and cargo and ran-gtp move to cytosol
3. ran binding protein and ran-gap promote: gtp hydrolysis, release of cargo, release of export receptor
4. empty export receptor returns to nucleus

Question 22

describe nfat (nuclear factor of activated t-cells) as an example of nuclear import and export regulation

Answer 22

high [ic ca2+] –> nuclear import
low [ic ca2+] –> nuclear export

import: calcineurin (protein phosphatase) removes pi from nfat, blocked NES and the lack of pi exposes the nuclear import signal, activation of gene transcription

export: the calcineurin leaves, atp + active protein kinase phosphorylates nfat and changes its conformation to expose the nes so it goes to cytosol

Question 23

describe an experiment that you can do to observe nfat transport (gated transport)

Answer 23

• express nfat-gfp fusion protein in t cells
• add calcium ionophore
• monitor fluorescence

Question 24

where is transmembrane transport involved it and what protein is needed

Answer 24

• er, mitochondria, plastids, peroxisomes
• protein translocators: transport of protein across membrane, protein usually unfolded

Question 25

how do proteins remain in cytosol

Answer 25

by association with hsp70 chaperones

Question 26

mitochondria and chloroplasts have their own genome and ribosomes but most of their proteins are nuclear encoded - where are these translated and what are its features

Answer 26

translated in cytosol, imported into organelle through post translational means and the protein is unfolded

Question 27

are proteins unfolded or folded when they go to mitochondria and chloroplasts through transmembrane transport

Answer 27

unfolded

Question 28

describe how to import proteins to the mitochondrial matrix

Answer 28

protein translocators: TOM (think of it as Translocase of Outer Membrane) and TIM23 (Translocase of Inner Membrane) complexes
- precursor protein has a mitochondrial signal sequence (peptide) with a specific n-terminal amphipathic a-helix
- it binds to receptor -> moves through TOM and TIM23 complexes into matrix space where the signal seq is cleaved
- proteins can also be further sorted

Question 29

describe how to import proteins to the chloroplast

Answer 29

protein translocators in chloroplast membranes
- precusor protein has a chloroplast signal seq with a specific n terminal amphipathic a helix
- signal seq cleaved in chloroplast
- different from mitochondrial signal seq for correct targeting in plants

Question 30

what is unmasked when chloroplast signal seq is cleaved

Answer 30

hydrophobic thylakoid signal sequence

Question 31

describe how to sort a protein into peroxisomes

Answer 31

• precusor protein has a peroxisomal targeting signal
• 3 aa at c-terminus (SKL)
• protein is post translationally folded
• transported across membrane by large translocator complex

Question 32

for the following destinations, state the mechanism, signal, and whether the protein is folded before transport:
i. nucleus
ii. mitochondria
iii. peroxisomes
iv. chloroplast

Answer 32

i. nucleus: gated, nls rich in lys and arg, folded before transport
ii. mitochondria: transmembrane, amphipathic a helix, not folded before transport
iii. peroxisomes: transmembrane, SKL at 3 terminus, folded before transport
iv. chloroplast: transmembrane, N-terminal amphipathic a helix, not folded before transport