SFP: cellular basis of protein synthesis and secretion

Question 1

describe how proteins are destined for translation in membrane-bound ribosomes

Answer 1

they have a targeting sequence in their N-terminus that direct the ribosome to the rough ER

Question 2

where does translation always begin

Answer 2

in the cytosol on free ribosomes

Question 3

the outer layer of the ER is continuous with __. the lumen of the ER is continuous with ___.

Answer 3

the nuclear envelope; perinuclear space

Question 4

list the basic steps involved in the secretory pathway

Answer 4

1. mRNA goes to a translating free ribosome
2. ribosome moves to the RER
3. protein goes through the golgi complex
4. protein enters a secretory vesicle
5. protein is released from the cell via exocytosis

Question 5

describe the function of the SRP?

Answer 5

a particle that binds to the signal sequence in the translating protein. it pauses translation of the protein and takes the whole protein-ribosome complex to the RER. the particle binds to the receptor, GTP converts to GDP, and the SRP is released.

Question 6

what is the translocon?

Answer 6

the channel in the RER membrane where the ribosome binds during translation

Question 7

once the ribosome and mRNA have been brought to the RER, where is the translating protein going?

Answer 7

into the lumen of the RER

Question 8

describe the process of forming single pass transmembrane proteins in the RER

Answer 8

• Internal signal sequence (ISS) binds to SRP and causes a pause in translation
• SRP binds to receptor, SRP releases, ribosome binds to translocon, ISS is inserted into the translocon
• Translocon releases ISS laterally into the membrane and translation continues. the N or C terminus will poke into the cytosol. The AA sequence in the ISS determines how the growing protein is oriented.

Question 9

describe the process of forming multipass transmembrane proteins in the RER

Answer 9

• 1st Internal signal sequence (ISS) binds to SRP and causes a pause in translation
• SRP binds to receptor, SRP releases, ribosome binds to translocon, 1st ISS is inserted into the translocon
• Translocon releases 1st ISS laterally into the membrane and translation continues
• 1st stop transfer sequence (STS) generated during translation pauses translation and releases STS into the membrane
• Translation resumes and 2nd ISS-SRP binds to the receptor, and a second loop in the membrane forms. This process of ISS and STS continues until the stop codon is reached and the protein is done.

Question 10

name the various types of protein modification in the RER

Answer 10

N linked glycosylation, adding GPI anchors, protein folding

Question 11

protein modification in RER: N linked glycosylation

Answer 11

oligosaccharide added to Asn in the lumen of RER. Occurs co-translationally. The oligosaccharide can be trimmed in the RER/golgi by various enzymes

Question 12

protein modification in RER: GPI anchors

Answer 12

GPI anchors made in the luminal side of the RER membrane get added to membrane-spanning domains near the C terminus of specific proteins. The amino group off ethanolamine will be used to link the GPI anchor and the c terminus of the protein

Question 13

where do GPI anchors in the membrane come from?

Answer 13

transported in vesicles from the RER that fuse with the plasma membrane

Question 14

protein modification in RER: BiP and calnexin/calreticulin

Answer 14

involved in protein folding

• BiP: Hsp70 that binds to the protein as it enters the RER lumen
• Calnexin and calreticulin: recognize oligo chains of N linked glycoproteins

Question 15

protein modification in RER: protein disulfide isomerase

Answer 15

enzyme in RER lumen that is important for correct protein folding by allowing for correct forming of disulfide bonds between cys residues; can break the bonds and let the correct ones reform

Question 16

describe ER associated degradation (ERAD)

Answer 16

misfolded proteins are transported from RER to cytosol for degradation via ubiquitin-proteasome

Question 17

describe the unfolded protein response

Answer 17

stress response from many unfolded proteins in the RER that activates a protein kinase pathway to stop translation

Question 18

what is the tubular vesicular network?

Answer 18

the intermediate between the RER and cis golgi

Question 19

what are the 4 main categories of protein processing in the golgi?

Answer 19

modifying N linked oligosaccharides, phosphorylating N linked oligosaccharides, O linked glycosylation, sulfation

Question 20

protein processing in Golgi: modifying N linked oligosaccharides

Answer 20

in the cis/medial cisternae, the golgi can add GlcNac, galactose, and sialic acid and remove mannose. It does this for plasma membrane and secreted proteins

Question 21

protein processing in Golgi: phosphorylation of N linked oligosaccharides

Answer 21

lysosomal enzymes get tagged with mannose-6-phosphate so they can be sorted into lysosomes. This happens in the cis Golgi network.

Question 22

protein processing in Golgi: O linked glycosylation

Answer 22

sugar added to ser, thr, or tyr one at a time in the cis and medial cisternae

Question 23

protein processing in Golgi: sulfation

Answer 23

sulfate added to tyr in the trans golgi network

Question 24

which lipids can be made in the golgi, and where are they made?

Answer 24

sphingomyelin and glycolipids; luminal surface

Question 25

what are the three secretory pathways in the golgi

Answer 25

constitutive (default pathway), regulated, and lysosome

Question 26

secretory pathway: constitutive

Answer 26

• clathrin coated vesicle pinches off the golgi
• clathrin coat dissociates
• vesicle fuses to the plasma membrane
• material in the vesicle ends up on the cytoplasmic side of the plasma membrane

Question 27

secretory pathway: regulated

Answer 27

• clathrin coated vesicle pinches off the golgi
• immature secretory granule forms (large vesicle)
• clathrin coat dissociates
• maturing secretory granule forms (bullseye appearance)
• mature secretory granule forms
• granule fuses and material releases through the membrane via exocytosis

Question 28

secretory pathway: lysosome

Answer 28

• clathrin coated vesicle pinches off the membrane
• clathrin coat dissociates
• vesicle enters the late endosome
• lysosome is formed

Question 29

what protein is needed to pinch off a vesicle? what is it?

Answer 29

dynamin; a GTPase

Question 30

describe anterograde vs retrograde vesicular trafficking

Answer 30

-Anterograde: COPII protein coated RER vesicles bring stuff to cis golgi network or TVN
-Retrograde: COPI protein coated vesicles can move anywhere back from the trans golgi; can go all the way back to the RER

Question 31

describe the basic process involved in sorting material into vesicles

Answer 31

• sorting sequence in protein binds to an adaptor protein
• adaptor protein found on inside of protein coat attaches the protein and the coat
• vesicle forms and moves to its destination

Question 32

name the three common coats for protein-coated vesicles and the types of cargo they’re associated with

Answer 32

• COPI coat: luminal RER or RER membrane proteins
• COPII coat: membrane and secreted proteins
• clathrin: lysosomal proteins

Question 33

briefly describe how exocytosis works

Answer 33

Target membrane has t SNAREs that bind to transport membrane v SNAREs. A RabGTP associated with the v SNARE hydrolyzes, allowing the two membranes to fuse.