MVU8 MEMBRANE PROTEINS - 3

Question 1

how can cellular membranes be made?

Answer 1

only be made by expanding pre existing membranes, not making new ones

Question 2

where are all proteins synthesised?

Answer 2

encoded by nuclear genes and trnalsate in the cytosol

Question 3

where is the sorting information for proteins, and when must proteins be sorted?

Answer 3

the sorting information is carried inside the proteins themselves
in the amino acid sequence
proteins must be sorted during or after translation to their correct location

Question 4

why are there many ribosomes around the ER membrane?

Answer 4

proteins are targeted to the ER co translationally
as they are being folded
secretory protein synthesis

Question 5

where are all secretory proteins inserted?

Answer 5

into or across the ER membrane
transported to further compartments

Question 6

what happens at the smooth ER?

Answer 6

no ribosomes, site of lipid synthesis

Question 7

what is the membrane of the ER made of?

Answer 7

continuous with the outer and inner nuclear membranes

Question 8

what are characteristics of targeting signals?

Answer 8

sequence within a protein
often independent of the structure or biochemical function of the protein
can be removed by proteolysis after targeting is complete, or remain part of native structure
recognised by a pattern, not exactly a specific sequence

Question 9

what are the steps of the targeting and delivery?

Answer 9

1. recognised signal on a protein
2. connect protein to the membrane
3. translocate protein into or across the membrane

Question 10

where do nascent polypeptides exit the ribosome? and what are the characteristics of that exit

Answer 10

through a tunnel in the large 60S units
tunnel is neutral, polar, too small for tertiary folding
surface around exit site provides binding sites for ER targeting mechanisms
30-40 AAs of nascent polypeptide between peptidyl transferase and exit

Question 11

what are characteristics of signal peptides?

Answer 11

hydrophobic central region of 8+ amino acids, with short polar regions on each side
in many cases the signal peptides are at the N terminus (at the beginning so that it goes to the ER right away)
shorter hydrophobic regions (8-16 residues)
often cleaved off after translocation

Question 12

what are characteristics of signal anchors?

Answer 12

signal peptides that also become TM helices
not cleaved off
can be in different placed in the protein
longer hydrophobic regions (18-24 residues)

Question 13

what are the targeting steps? (more detailed)

Answer 13

1. recognise signal on newly translated protein
   ribosomes translate protein with signal
   signal recognition protein (SRP) is soluble protein that binds signal and ribosome during translation, slows down translation
2. connect protein to the membrane
   SRP receptor (SRP-R) is membrane protein that binds the ribosome-SRP complex
   SRP-R links ribosome to translocon pore in ER
3. translocate protein into or across the membrane
   energy of translation on ribosome drives polypeptide through the translocon

Question 14

what is the structure of the SRP?

Answer 14

ribonucleoprotein (RNA+protein): 6 protein subunits and 1 RNA
signal sequence recognition subunit with GTPase activity (stimulates GTPase activity of receptor)
translation regulatory domain at opposite end (pauses ribosome)
RNA strand forms flexible linker

Question 15

what happens after SRP binds ribosome?

Answer 15

SRP pauses translation and binds GTP
ribosome-SRP complex binds to the SRP-R on ER
ribosome moves to the translocon and becomes tightly bound
SRP and SRP-R dissociate from ribosome
translation resumes and polypeptide goes to the lumen
lumenal polypeptides do not touch the cytoplasm

Question 16

where does GTP play a role in all this?

Answer 16

SRP attached to ribosome is in a GTP bound state
SRPR is also attached to GTP when it recognises SRP-ribosome complex
GTP hydrolysis by both SRP and SRP-R
dissociate and recycle
GTP is used like a switch

Question 17

how does the ER translocon work?

Answer 17

called the sec61 complex
2 parts that form both sides of aqueous pore
inactive pore is plugged by a part of the protein
the active pore is open but tightly sealed onto ribosome
signal polypeptide pushes the plug away
inside of the pore it is polar neutral
the 2 parts of the pore open laterally to integrate TM helices into membrane

Question 18

how does the integration of TM helix work?

Answer 18

protein with N terminal signal sequence and TM helix:
1. signal peptide starts translocation of lumenal part
2. TM helix is recognised by translocon and integrated laterally into membrane during translation
3. cytosolic part if translated in cytosol

Question 19

what are type 1 TM proteins?

Answer 19

N terminus in lumen
C terminus in cytosol

Question 20

how does the signal anchor integration work?

Answer 20

1. signal anchor opens translocon like a signal peptide
2. translocon recognises charges next to signal anchor to determine orientation in membrane (pos in cytosol, neg in lumen)
3. signal anchor is recognised as a TM domain and integrated laterally

Question 21

what is the signal anchor?

Answer 21

signal anchor is a signal peptide with long hydrophobic region that is not cleaved off but becomes the TM domain
length of the hydrophobic region is 18-24 AA long

Question 22

what are type type 2 TM proteins?

Answer 22

N terminus in cytosol
C terminus in lumen
signal will be a few AAs before the N terminus

Question 23

what are multi pass TM proteins?

Answer 23

combinations of signal anchor and TM helices causing alternating orientation of TM proteins

Question 24

how can the topology (TM organisation) of secretory pathway proteins be predicted?

Answer 24

hydrophobicity: number of TM helices
charge distribution: orientation in the membrane
can then predict other PT modifications

Question 25

what does the attaching of glycans to secretory proteins do?

Answer 25

helps stabilise the native state
protect against proteases
fuctions in cell surface signaling

Question 26

what is the motif that is around the AA that is attached to the glycan?

Answer 26

Asn-X-Ser/Thr motif
glycan attaches to asparagine

Question 27

what is the structure of the glycan

Answer 27

it is always the same
mostly mannose with 3 glucose
the first part is made of 2 N-acetylglycosamines

Question 28

what enzyme performs the N linked glycosylation?

Answer 28

oligosaccharyl transferase (OST)
acts on most motifs in the lumen, depending on the accessibility

Question 29

how can glycans be altered after addition?

Answer 29

can be modified but not removed till the degradation of the protein

Question 30

what is the glycosylation process?

Answer 30

1. oligosaccharides are synthesised attached to a specialised lipid in the ER
2. OST attaches glycan during translocation
3. state of glycan is used as a signal in ER quality control of folding
4. glycan is modified in golgi after exit from ER
   the glycan is the signal that the protein belongs to the secretory pathway