Professor Uglade Lecture 9 (membrane proteins 3)

Question 1

Where are proteins encoded and translated?

Answer 1

all proteins are encoded by nuclear genes and translated in the cytosol (all proteins encoded by nucleus of the cell)

Question 2

How can cellular membranes be made?

Answer 2

they can only be made by expanding pre-exisiting membranes

Question 3

How are proteins sorted to their correct compartment?

Answer 3

proteins must be sorted during or after translation to their correct compartment or membrane
-sorting info is carried inside the proteins

Question 4

Where are secretory pathway proteins inserted?
And what parts of the membranes are continious?

Answer 4

they are inserted into or across the ER membrane
-outer and inner membrane are continuous with ER

Question 5

Where are secretory pathway proteins transported?

Answer 5

-transported to further compartments-Golgi, PM, endosomes, lysosomes

Question 6

Difference between Rough ER and Smooth ER?

Answer 6

-Rough ER- many attached ribosomes, secretory protein synthesis
-smooth ER:no ribsomes, sites of lipid synthesis

Question 7

What do targeting signals specify?

Answer 7

-they are sequences within a protein that specify its organelle localization (zip code) or signal peptide

Question 8

How are targeting signals removed, and how are they related to the protein structure?

Answer 8

-they may be removed by proteolysis after the targeting is complete, or form part of the native structure
-they are often independent from the structure or biochemical function of the protein

Question 9

How are targeting signals recognized?

Answer 9

-they are recognized by their pattern but usually not the exact sequence

Question 10

What are the 3 targeting steps (in targeting signals)

Answer 10

1. recognize a signal on a protein
2. connect protein to the membrane (of the ER in this case)
3. translocate protein into or across the membrane (if it is a lumenal protein)

Question 11

When do secretory proteins enter the ER?

Answer 11

During the translation of their mRNAs

Question 12

What is the signal hypothesis?

Answer 12

-extra sequence is a targeting signal peptide whose main function is to direct insertion into ER

Question 13

How do signal peptides start the signal mechanism, and when is the signal peptide cleaved?

Answer 13

signal peptide must start mechanism to connect ribosomes to translocation pore
-signal peptide is cleaved off after targetin is finished

Question 14

How do nascent polypeptides exit the ribosomes?

Answer 14

-through a tunnel in the large 60s subunit

Question 15

what are the properties of the ribosomes exit tunnel?

Answer 15

-tunnel is neutral, polar, too small for tetiary folding (just a line of polypeptides)

Question 16

what does the surface of the exit site in ribosome exit tunnel provide, and how many amino acids are between the transferase site and the exit?

Answer 16

surface around the exit site provides binding sites for ER targeting mechanisms
-30-40 amino acids of nascent polypeptide between peptidyl-transferase site and the exit

Question 17

What do secretory signal peptides direct/do?

Answer 17

-they direct proteins to the ER for translocation into or across the membrane through cotranslational mechanism (mostly)

Question 18

How are secretory pathway proteins directed into organelles

Answer 18

-they have additional targeting signals such as a polypeptide motif (sequence pattern)
-sometimes a post translational modification

Question 19

What do organelles with no secretory pathway do, and what are examples?

Answer 19

-they have their own tareting signals
-ex: mitochondria, nuclei

Question 20

What is the signal polypeptide pattern composed of?

Answer 20

-hydrophobic central region 8 or more residues long, with short polar regions on each side

Question 21

what do polar regions of signal polypeptide pattern determine?

Answer 21

-they determine the direction of which end the protein is inserted

Question 22

Where are signal peptides normally found, and when are they cleaved?

Answer 22

-signal polypeptides are at the N-terminus
-shorter hydrophobic regions (8-16 residues)
-often cleaved off after translocation (cleaved off by peptidases)

Question 23

What are signal anchors and how do they differ from signal peptides?

Answer 23

-they are signal peptides that also become TM helices
-unlike signal peptides, signal anchors are not cleaved off, and they have a longer hydrophobic region (18-24 residues, since they have to be across the whole membrane
-they can also be in diff places in the protein

Question 24

How is a signal recognized on a newly translated protein, and what does SRP do?

Answer 24

ribosome begins translating polypeptide with a signal, signal recognition protein (SRP) which is a soluble protein that binds signal and ribosome during translation

Question 25

How is a protein connected to a membrane?

Answer 25

SRP-receptor (SRP-R) is a membrane protein that vinds the ribosome-SRP complex (has receptor in membrane of ER)

Question 26

What does the SRP-R link?

Answer 26

SRP-R links ribosomes to translocon pore in ER (where ribosomes attach, the ribosome pushes nascent peptide into ER)

Question 27

How are proteins translocated across the membrane?

Answer 27

The energy of translation on ribosome drives polypeptide through the translocon

Question 28

What is a signal receptor particle (SRP) composed of?

Answer 28

6 protein subunits and 1 RNA (macromolecule of RNA and proteins)

Question 29

What activity does SRP do?

Answer 29

it is a signal sequence recogition subunit with GTPase activity

Question 30

where is the translation regulatory domain on the SRP, and what does the RNA strand form?

Answer 30

-translation regulatory domain at the opposite end -RNA strand form a flexible linker

Question 31

What does SRP sample?

Answer 31

SRP samples all nascent polypeptides that emerge from ribosomes

Question 32

Whena signal is recognized what does SRP do?

Answer 32

it attaches tightly to both the signal and the ribosome, it pauses translation at the ribosome and binds GTP

Question 33

what causes the ribosome do stop translation?

Answer 33

-when the hydrophobic region is recognized by the SRP there is a conformational change, so that translation stops and it can get to the ER membrane

Question 34

After ribosome translation is stopped, what does the ribosome SRP complex do?

Answer 34

it binds to the SRP-R on ER

Question 35

After ribosome-SRP is bound to SRP-R on the ER what occurs?

Answer 35

ribosome moves to the translocon and becomes tightly bound

Question 36

after translocon is tightly bound, what happens to SRP and SRP-R, and what 2 steps occur after this?

Answer 36

SRP and SRP-R dissociate from ribosome, and translation resumes and polypeptide translocates into lumen -lumenal polypeptide does not contact the cytosol

Question 36

after translocon is tightly bound, what happens to SRP and SRP-R, and what 2 steps occur after this?

Answer 36

SRP and SRP-R dissociate from ribosome, and translation resumes and polypeptide translocates into lumen -lumenal polypeptide does not contact the cytosol

Question 37

How is SRP attached to ribosomes ,and how is SRP-R bound to SRP-ribosomes?

Answer 37

SRP is attached in the GTP bound state -SRP-R is also in GTPase, and is in GTP-bound state when it recognizes SRP-ribosomes

Question 38

What dissociates SRP and SRP-R from GTP- bound state? What is GTP like in this context?

Answer 38

GTP hydrolysis by both SRP and SRP-R dissociate and recycle them -GTP is used like a switch

Question 39

What does the Sec61 (ER translocon) form?

Answer 39

-it has 2 parts that form both sides of aqueous pore (open as a door in the membrane)

Question 40

What is the inactive Sec61 pore plugged by, and what are the properties of the active pore?

Answer 40

-inactive pore is plugged by part of a protein -active pore is open but tightly sealed onto ribosome (pore only allows passing of nascent peptide) -inside pore is neutral, polar

Question 41

how do the 2 parts of the Sec61 (ER translocon complex) open

Answer 41

the 2 parts of the pore open laterally to integrate TM helices into membrane

Question 42

How does translocation of lumenal protein occur?

Answer 42

-the signal peptide triggers opening of the translocon

Question 43

in what state are polypeptides translocated, and how is the movement driven?

Answer 43

translocated in an extended, unfolded state -movement of polypeptide is driven by energy of translation pushing it out of the ribosome

Question 44

What removes signal peptides during translocation of lumenal proteins?

Answer 44

signal peptidase often removed signal peptide during translocation (not sequence specific but has a preferred site)

Question 45

How is a protein integrated into TM helix?

Answer 45

-protein starts with N-terminal sequence and TM helix

Question 46

What does the signal peptide do and how is the TM helix integrated in the integration of TM helix? Where is the cytosolic part translated?

Answer 46

-signal peptide start stranslocation of lumenal part -TM helix is recognized by translocon and integrated laterally into membrane during translation -cytosolic part is translated in cytosol

Question 47

what is a type 1 TM protein?

Answer 47

Type 1 TM protein has N-terminus in lumen, C-terminus in cytosol

Question 48

What is a signal anchor in composition, and what does it become?

Answer 48

it is a signal pepide with long hydrophobic regions that is not cleaved off -it becomes the TM domain-length of hydrophobic region is 18-24 amino acids

Question 49

What are the steps for signal anchor integration? 1. What does it open?

Answer 49

1. signal anchor opens translocon like a signal peptide

Question 50

what is step 2 for signal anchor integration? 2.what does translocon recognize? Where are the charges

Answer 50

2.translocon recognzies charges next to the signal anchor to determine orientation in membrane -positice charges in cytosol, negative charges in lumen

Question 51

Compare and contrast SRP (signal sequence, signal anchor) and HSP70 1. WHat do they recognize in a polypeptide 2. will they compete for binding to the same sites 3. how is binding regulated

Answer 51

1. They both recognize hydrophobic residues 2.they dont compete for biniding sites since SRP recognizes a stretch of amino acids that is in the n-terminus and HSP70 will recignize hydrophobic patches 3. HSP70--> regulated by HSP40 bringing in the substrate and binding it tightly to ADP whereas SRP--> regulated by GTP