MCB 110 Final Exam Updated Flashcards

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What does Ran do? Is it a GTPase or ATPase?

Nuclear import/export gradient (Ran-GDP cytosol, Ran-GTP in the nucleus). It is a GTPase

What dose Sar1 do? What does it use for the inner and outer layers? Is it a GTPase or ATPase?

COPII coat assembly (ER to Golgi)

Sec 23/24 for the inner layer and Sec 13/31 for the outer (note that Sec 23/24 is first, so it must be the inner layer)

It is a GTPase

What does Arf1 do? Is it a GTPase or ATPase?

COPI coat and clathrin coat assembly

It is a GTPase

What does dynamin do? Is it a GTPase or ATPase?

Vesicle scission (pinching off membrane)

It is a GTPase

What does SRP and its receptor do? Is it a GTPase or ATPase?

Binding and handoff to the Sec61 translocon. Its’s used for proteins to be sent to the ER

They are both GTPases (they hydrolyze each others’ GTPs actually)

What does Ef-Tu do? Is it a GTPase or ATPase?

It brings charged tRNAs to the ribosome

It is a GTPase

What does EF-G do? Is it a GTPase or ATPase?

Ribosome translocation (it actually takes two steps, tRNAs spontaneously shift then you have EF-G)

It is a GTPase

What do Hsp70/BiP/Hsc70 do? Is it a GTPase or ATPase?

These are chaperones that bind hydrophobic patches. Hsc70 specifically coats clathrin vesicles.

These are ATPases

What does the proteosome do? What are the two components, and which of those are ATPases/GTPases?

It is used for protein degradation. The 19S is the ATPase that unfolds the substrate protein and feeds it into the 20S barrel. It also has Rpn11 to remove the poly Ub tail. The 20S subunit is the barrel where the substrate protein is degraded

What does NSF do? Is it a GTPase or ATPase?

It untangles and recycles SNARE complexes after vesicle fusion. It is an ATPase

How do t-SNAREs and v-SNAREs interact? What complex do they form and how does it fuse membranes?

They form a four helix bundle and then zipper-like action brings membranes very close together to fuse

What does amino-acyl tRNA synthetases (aaRS) do? Is it a GTPase or ATPase?

Charge tRNAs with an activated amino acid formed by hydrolyzing ATP to AMP (so yes it’s an ATPase). Note that the editing site cleaves incorrectly charged amino acids or even incorrectly activated amino acids via size exclusion. But when you edit, no more ATP is burned (so no matter what in the whole thing, you just burn 1 ATP)

What does V-ATPase do?

Pumps H+ to acidify lysosomes and endosomes

What does Ub E1 enzyme do? Is it a GTPase or ATPase?

It activates Ub to start the degradation cascade by binding Ub to the Cys of E1. It is an ATPase

For COPII, indicate the direction of transport and the key players involved

ER to Golgi (anterograde)

Sar1 GTPase (activated with Sar1-GEF), inner coat: Sec 23/24, outer coat: Sec 13/31

For COPI, indicate the direction of transport and the key players involved (also what’s the retrieval signal).

Arf1 GTPase, retrieval signal: KDEL

The cell uses clathrin to ship proteins from where to where (there are two of these)? What’s the GTPase involved?

Trans golgi to endosome/lysosome OR plasma membrane to endosome (this is endocytosis)

The key player is Arf1 for the endosome, but for endocytosis, PIP2 activates AP2 which recruits clathrin

What is the lysosomal targeting singal?

M6P. M6P receptors drop their cargo in the endosome due to low pH via a conformational change.

Describe the Ran gradient and the function of FG repeats.

In nuclear transport, the cytosol has high Ran-GDP due to Ran-GAPs, while the nucleus has high Ran-GTP due to Ran-GERF. Importins bind to cargo and then pass through the pore into the nucleus. Then, Ran-GTP comes in to displace the importin (mutually exclusive binding). For exportins, both the exportin and Ran-GTP bind to the cargo (cooperative binding) and then they leave the nucleus. Once in the cytosol, Ran-GAP hydrolyzes the GTP to release the cargo.

FG repeats block large proteins from diffusing through the nuclear pores and they create a gel-like phase. Small metabolites < 1 kDa can still pass through though.

Describe the eukaryotic closed loop in mRNA and the proteins that bind to it and how translation starts.

eIF4E binds the 5’ cap, DABP binds the poly A tail, and eIF4G connects eIF4E and PABP. The Pre-Initiation Complex (PIC) scans down from the 5’ cap and searches for the Kozak sequence to find the start codon. Eukaryotes also use normal Met-tRNA, but remember, the initiator tRNA is different from the tRNA used in regular elongation for Met

Describe how translation is initiated in bacteria.

Bacteria use fMet-tRNA (formylated Met), and the mRNA Shine-Dalgarno sequence base pairs with the ribosome’s small subunit’s 16S rRNA to align the ribosome with AUG at the P site. Note that IF2 brings fMet-tRNA to the P site, and IF1 and IF3 block the E and A sites to force the tRNA to go to the P site.

Ub attaches to what amino acid on target proteins to signal degradation? Also describe what X48 and X63 proteins go to, where X is the amino acid you identified.

Lysine

K48 goes to the proteosome, while K63 goes to the lysosome/DNA repair/endocytosis

Describe the Ub cascade and how many unique enzymes there are for each. For the third one, describe the two different types of it.

E1 (activating): Use ATP to form Ub0Cys-E1 (only 1 to 2 E1 enzymes)

E2 (conjugating): carries the activated Ub in the form Ub-Cys-E2 (about 35-40 enzymes)

E3 (ligase) (hundreds of enzymes)

• HECT E3 forms a covalent intermediate with Ub then transfers it to the target protein (Remember, the C in HECT means Covalent intermediate)

• RING E3 doesn’t form a covalent intermediate and instead brings E2 and the target close together and the exchange happens there

Describe the Unfolded Protein Response (UPR)

A misfolded protein binds to IRE1, which gets activated via autophosphorylation. Then, IRE1 cleaves XBP1 in mRNA, which then leads to translation of XBP1 mRNA. Then, this leads to nuclear import of a transcription factor, which causes overexpression of chaperones.

In short, IRE1 gets activated to ultimately produce more chaperones to fold the unfolded proteins, but this is the big picture takeaway. You must know the details.

What is the ER signal sequence?

About 15 - 30 amino acids with a stretch of nonpolar residues

How many tRNAs are there total and why?

About 40 - 50. You don’t need 61 (64 - 3 stop codons) because of the wobble pair with the 1st anti codon pair on the 5’ end

How many amino acids can fit in the ribosome exit tunnel?

30 - 40

Does the RNA or protein in the ribosome do the catalysis?

RNA

What is the most evolutionary conserved phase of translation?

Elongation (similar mechanism in prokaryotes and eukaryotes)

What class of RFs mimic tRNA at the A site and what does this do?

Class I RFs. This cleaves off the peptide chain with tRNA using H2O as the nucleophile

Why did Anfinsen’s experiment use dilute protein concentration? How does this compared to the cytosol?

In the cytosol, the protein concentration is high, so intramolecular hydrophobic interactions are frequent, which can cause protein aggregation. That’s why Anfinsen’s experiment had low concentration.

RNAse A folds, but it also does two things. Hint cleavage of something and formation of something else. What are they?

Cleavage of its signal sequence and forms disulfide bonds

Proteins at the ER get shipped where?

To the golgi

What are three things that are sent from the golgi to the ER?

Cargo receptors, lipids, and SNAREs. Generally, any ER proteins the ER needs

What is the mitochondria signal sequence?

Positively charged and has a amphipathic helix

What is one similarity between the ER and mitochondria signals and one difference?

Both signals are cleaved, while the ER signal forms a loop-like structure during translocation but mitochondria don’t

Describe Hsp70 when ATP bound vs not ATP bound.

ATP bound: peptide-binding pocket releases substrate protein (lid is open). Or the interactions with the substrate is weak

ADP bound: the lid closes, and the pocket tightly binds the protein and folds it

The mitochondrial Hsp70 binds to the protein imported by what complex? What does it also do to do this?

TIM23 complex. It burns ATP, prevents backsliding, and provides the driving force to do this

Lysosome V-ATPase pumps H+ into or out of the lysosome?

Into

Why can’t lysosomal hydrolases degrade in the lysosomal membrane?

Because of the presence of a thick glycon layer (surprisingly, it’s not because of the pH)

When does M6P modification of lysosomal hydrolases occur?

During transport through Golgi (think about it, the sugar gets added as the protein goes through Sec61)

What does the ESCRT complex do and form?

They mediate budding of membranes for the formation of MVBs, and they mediate pinching off the membrane vesicles inwardly in endosomes. In general, it pushes a membrane away from the cytosol into an internal space (opposite of COPII or clathrin)

TIM23 uses a GTPase or ATPase? Also, there is another driving force. What is that?

ATPase. It’s also driven by the membrane potential across the mitochondrial inner membrane

A general GTPase-GTP binds to what general class of molecules?

Effector

What do Rab-GTPases (active form) bind to?

Tethering complexes. This tethers the vesicle with the target membrane

What does the J protein do?

It delivers the substrate protein and causes Hsp70 to hydrolyze ATP to ADP

What are JAMM and USP examples of (general class of enzymes)? What do they do?

These are DUBs (De-Ubiquinating Enzymes) that remove poly Ub tails and save proteins from degradation

Something can still pass through FG repeats spontaneously. What is it?

Small metabolites (large proteins that fit through the pore can still pass through)

Where does 4E-BP1 bind to on mRNA? What is the activator that inhibits this protein?

5’ cap. mTORC1 inhibits 4E-BP1

What is the amino acid signal sequence for nuclear export?

PKKKRKV

Where is the MTS located (C-terminal or N-terminal), and if you block that terminal, what happens?

N-terminal. If you block it (like with a fluorescent protein), it wont’ go to the mitochondria

Where does Maskin bind on the mRNA? What can displace it?

The 3’ poly A tail. PABP can displace it when the poly A tail grows big enough