Intracellular Compartments and Protein Sorting

Question 1

Where are the proteins for organelles made?

Answer 1

They are coming from the cytoplasm - some will be shipped to the ER, some will be imported to the nucleus, and some to the Golgi

Question 2

How do proteins get directed to the correct cell address?

Answer 2

• signal sequences and sorting receptors (information about where the protein is supposed to go is in the sequence of the protein itself
• signal sequence has to bind to the receptor and the receptor tells the protein where it needs to go

Question 3

what happens if a protein has no signal sequence?

Answer 3

it stays in the cytoplasm

Question 4

what are the two types of signal sequencing of a protein?

Answer 4

• continuous (at the N-terminus)

- discontinuous (signal patches)

Question 5

What are the three main mechanisms of protein trafficking?

Answer 5

1. gated (nuclear pores)
2. transmembrane (translocation - unfolded proteins move through trasnlocator to different topological space)
3. vesicular (moves through topologically equivalent space)

Question 6

What is GATED transport?

Answer 6

bidirectional transport that transports between cytoplasm and nucleus

Question 7

what has to move through a nuclear pore?

Answer 7

any proteins associated with the genome (proteins and enzymes associated with DNA replication and transcription)

Question 8

where are ribosomal subunits assembled before it is exported to the nucleus?

Answer 8

the nucleolus is where ribosomal RNA is transcribed

Question 9

what are 2 types of transport through pores?

Answer 9

1. Passive (small, water soluble molecules

2. Active-Transport (for folded proteins, RNA and ribosomal subunits

Question 10

What are 3 things required to move nuclear proteins to the nucleus?

Answer 10

1. sorting signa (NLS)l/exporting signal
2. receptor (interacts with nuclear pore)
3. energy(GTPase)- (needed to move cargo from low concentration to high concentration)

Question 11

what are sorting signal sequences (NLS)?

Answer 11

sequence anywhere on the protein that is rich in + charged amino acids of arginine and lysine

Question 12

What are nuclear import receptors (importins)?

Answer 12

nuclear import receptors recognize and bing NLS. They can also bind to FG repeats in the nuclear pore complex (NPC)

• different types of importins bind a subset of cargo with a specific NLS
• SHAPE MATTERS*

Question 13

TRUE or FALSE: importins and exportins can move through pores with or with our cargo

Answer 13

TRUE

Question 14

what type of energy is required for cytoplasm-nucleus transport?

Answer 14

GTP Hydrolysis - linked to a concentration gradient and can be hydrolyzed to form GDP

Question 15

What are GTPases (or GTP-binding proteins)

Answer 15

GTPase are a family of proteins that can act as a molecular switch - they are active when binding to GTP and inactive when they are hydrolyzed to form GDP
-the main protein is RAN

Question 16

What are GTPase Activating Proteins (GAPS)

Answer 16

• GAPS are helpers that interact with G proteins to make hydrolysis of GTP move fast
• they also help G proteins turn off

Question 17

What are Guanine Nucleotide Exchange Protein (GEFS)

Answer 17

GEFS help speed up the reaction of the G protein with removing GDP and replacing it with GTP

Question 18

What are the two conformational changes of RAN GTPase?

Answer 18

1. GTP-bound (Ran-GTP; nucleus)

2. GDP-bound (Ran-GDP; cytoplasm)

Question 19

what way does RAN move in?

Answer 19

RAN moves from the nucleus (GTP) to the cytoplasm (GDP)

Question 20

What is transmembrane transport?

Answer 20

Transmembrane transport is the transport of unfolded proteins from the cytosol to a different topological space (plastids, mitochondria, ER, and peroxisomes) through a translocator
- this type of transport can be co-translational or post-post-trasnlational

Question 21

What 5 protein complexes are involved in mitochondrial protein translocation

Answer 21

TOM –> can move proteins into intercellular space
SAM –> involved in folding of proteins for the membrane (beta barrels - porins)
TIM22 –> helps fold inner membrane proteins
TIM23 –> used for proteins going into the matrix
OXA –> for inner membrane proteins coming from the matrix

Question 22

What are 3 things required for import from cytosol to mitochondria?

Answer 22

1. signal sequence (can be at N-terminus or internal of protein)
2. receptor (protein translocator complex in membrane)
3. energy (ATP and proton gradient,)

Question 23

What is the structure of a signal sequence for matrix proteins?

Answer 23

the signal sequence is an amphiphilic alpha hell with charged, polar amino acids on one side and unchanged, hydrophobic amino acids on the other

Question 24

What 2 protein trasnlocators recognize the signal sequence?

Answer 24

• TOM

- TIM

Question 25

what is the difference between the TOM and TIM complexes?

Answer 25

- TOM (trasnlocase of the outer membrane) transfers all proteins across the outer membrane - TI (trasnlocase on the outer membrane) transfers all proteins across thinner membrane

Question 26

What does the chaperon HSP70 help with?

Answer 26

-In the cytoplasm as proteins are being picked up and translated, chaperons come in and help. Typically chaperons help proteins fold but in this case, the HSP70 keeps the protein unfolded

Question 27

How do ATP hydrolysis and membrane potential drive transport of proteins into the matrix?

Answer 27

- ATP hydrolysis releases cytosolic chaperons (HSP70) - positively charged amino acids of signal sequence are electrophoresed across the inner membrane - Mitochondrial HSP 70 pulls protein into matrix - ATP hydrolysis drives confirmation change to release protein

Question 28

is transmembrane transport into chloroplasts post-translational or co-translational?

Answer 28

post-translational: both proteins complete translation in the cytoplasm prior to being imported

Question 29

What is the difference between mitochondria and bacteria when it comes to interrogating porins into the outer membrane

Answer 29

- they share similar processes; the only difference is that mitochondria uses SAM complex and bacteria uses BAM complex

Question 30

what are the 3 major functions of the ER

Answer 30

1. protein synthesis - for endomembrane system, plasma membrane and secreted proteins 2. Lipid synthesis (for all organelles) 3. Ca2+ storage

Question 31

what is the difference between rough ER and smooth ER

Answer 31

- The rough ER is where ribosomes are located (for translation) - The smooth ER is where lipid synthesis occurs

Question 32

What 3 things does co-translational ER protein import require?

Answer 32

1. Signal sequence - 16-50 aa sequence at N-terminus, and 8 or more hydrophobic aa in the middle 2. Signal receptor - Signal Recognition Particle (SRP) soluble riboprotein complex with hydrophobic pocket to bind signal 3. Energy - GTP (in mammals) and ATP (in yeast)

Question 33

For transmembrane translation into the ER, the signal receptor particle (SRP) not only binds to the growing peptide, but also binds to the ribosome itself. Why is this important?

Answer 33

This process is co-translation; it is important that SRP binds to the ribosome as well because it can pause translation (translational pause domain) and prevent it from finishing until ribosome is docked to the ER membrane

Question 34

What does the hinge domain allow the SRP molecule to do?

Answer 34

-The hinge domain allows the complex to bend around the ribosome when it first binds the signal sequence

Question 35

What are the two orientations of single-pass transmembrane proteins with internal signal sequence

Answer 35

1. N-terminal in ER lumen--> when + charged aa are on the side of C-terminus side 2. C-terminal in ER lumen--> when + charged aa are on the side of N-terminus

Question 36

What do multi-pass transmembrane proteins have that single-pass don't?

Answer 36

Multipass transmembrane proteins will have a series of internal start transfer and stop transfer sequences that will dictate which portions of the protein remain embedded in the membrane and which will be found in the cytosolic and lumen side of the membrane