Ch 12 Pt 2

Question 1

The ER is organized into a

Answer 1

netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol

Question 2

The ER and nuclear membranes form a

Answer 2

continuous sheet enclosing a single internal space, ER lumen

Question 3

SER is the primary site of

Answer 3

ER vesicle budding, transitional zones.

Question 4

In specialized cells the SER is involved in

Answer 4

lipid metabolism, and steroid biosynthesis.

Site of detoxification (ex. liver has lots of smooth ER) Sarcoplasmic reticulum - smooth ER ca2+ storage center

Question 5

SER has a network of

Answer 5

Network of tubules

Question 6

RER is the primary site of

Answer 6

protein synthesis of integral transmembrane proteins (all transmembrane made from rough ER )

, as well as “soluble” secreted proteins.

Secreted protine (soluble) made in ER - then in vessicles to out of er

Question 7

RER is associated with

Answer 7

ribosomes

Question 8

ER breaks into fragments, fragments reseal to form closed vesicles called

Answer 8

microsomes: small version of ER, retain functionality of ER

can study as if is really the ER

Question 9

Smooth vs Rough microsomes

Answer 9

Rough - more dense - further down.
can use a density (ex. sugar) at right density float vs sink

Question 10

G. Blobel and Dobberstein, 1975 experiment did what

Answer 10

mRNA encoding for the secretory IgG light chain of secreted antibody is translated in an in vitro translation system in the….
Absence of microsomes
Presence of microsomes.

Question 11

Blobel’s experiment showed

Answer 11

SDS page analysis shows that translation in the presences of microsomes results in a slightly smaller polypeptide

in the presences of microsomes, a leader sequence of the precursor protein directs it to the ER where it is cleaved off by a signal peptidase

Question 12

if, in Blobel’s experiment, it was a soluble cytosolic protein being looked at what would the results be

Answer 12

it would be the same size on the SDS page because it is not transported into the ER for cleavage

Question 13

Are secreted proteins immediately secreted out of the cell after ribosomes synthesize them in the cytosol

Answer 13

No, They go to the ER

Question 14

Rough microsomes produce proteins. Trypsin (protein digesting enzyme) rarely enters the microsome. Would you expect a secreted protein to be digested or not digested by Trypsin?

Answer 14

No, because the protein is inside of the rough ER (microsome) where the enzyme cannot get to it

Question 15

Translation-translocation in vitro system consists of isolated mRNA, ribosomes, microsomes. Mostly short chain IgG light chains produced. If complete, oversized light chains added AFTER microsome, signal sequence is not cleaved. WHY?

Answer 15

Microsomes must be added before the first 70 amino acids are polymerized in order for proteins to be processed in the microsome. Proteins must be translated during translation (co translational)

The removal of the signal sequence occurs during translation, not after

Question 16

Co translational import

Answer 16

translation occurs while translocated
signal sequence associates with binding site
ribosomes associate with er membrane
signal peptidase, associated with translocator, cleaves as being translated

Question 17

Two types of proteins made in the ER

Answer 17

Membrane associated
soluble

Question 18

ER Signal Sequence Directs ____ to ER membrane

Has ___ at its center

Binds to ___

Answer 18

transmembrane and water soluble proteins to ER membrane

Has** eight or more nonpolar amino acids **at its center

Signal sequence varies in amino acid sequence

Binds to: SRP

Question 19

SRP

in ___

___ lined with ____

Answer 19

(Signal Recognition Particle)
rodlike ribonucleoprotein
(six polypeptides and one RNA)

in cytosol

Large hydrophobic pocket lined with Methionines

Question 20

Ribosomes:

Answer 20

Macromolecular complex of RNA and protein. Consists of a large and small subunit

Question 21

SRP wraps around

Answer 21

large subunit: One end binds to signal sequence and the other end blocks the elongation factor binding site (between large and small ribosomal subunits) This halts protein synthesis

Question 22

Why would you want SRP to pause protein translation

Answer 22

Gives protein time to get to the ER (not want to be fully made before get to ER)

Question 23

SRP binds to ER sequence and SRP receptor steps

Answer 23

SRP binds to signal sequence
(emerging from ribosome)

Conformational change in SRP
exposes a binding site for SRP
receptor (on ER membrane)

SRP-ribosome complex is brought
to unoccupied translocator

Question 24

Free VS membrane bound polyribosomes

Answer 24

Ribosome is not fixed to the ER

Question 25

polyribosomes

Answer 25

many ribosomes translating mRNA at the same time - allowing multiple copies of protein to be made at the same time

Question 26

Sec 61 is a ___ that forms ___ It is the ___ ___ gating

Answer 26

heterotrimer (three subunits) that forms a dynamic gated channel. It is the core of the translocator it opens and allows the polypeptide to pass through is gated with a plug lateral gating - opens sideways into bilayer ("packman") signal peptide with hydrophobic regions is released into the bilayer (bound or degraded)

Question 27

Ribosome is present when ___ is open during translocation of a soluble protein across the ER

Answer 27

Sec 61

Question 28

Steps of the Translocation of a Soluble (Lumenal- Secreted) Protein Across the ER

Answer 28

1. ER signal sequence binds to a specific binding site in the pore, serves as a start-transfer signal that opens the pore 2. Signal sequence remains bound to Sec 61 complex during translocation 3. Signal sequence also in contact with lateral seam (lateral gating) 4. Signal peptidase cleaves signal sequence, mature protein released into lumen.

Question 29

Single Pass TM protein

Answer 29

N-terminal signal sequence opens translocator. Additional hydrophobic segment (stop transfer sequence), interacts with binding site within the pore Translocator opens at the seam (lateral), signal sequence cleaved and mature protein is released laterally into bilayer (Stop transfer sequence anchors protein) N terminal - Lumen C terminal - Cytosol

Question 30

If a single pass TM protein was transported to the plasma membrane where would the N/C terminal be

Answer 30

N terminal - extracellular

Question 31

Single Pass TM protein when N terminus-Cytosol

Answer 31

Internal Start-transfer sequence Positively-charged aa cluster, upstream of the Start-transfer sequence (NH2-side). Causes this region to remain on the cytoplasmic side, while hydrophobic start-transfer sequence and C-terminus passed across membrane.

Question 32

If pos charged AA follow the start transfer sequence

Answer 32

the orientation is the opposite (N- terminal-lumen side)

Question 33

Multipass TM protein

Answer 33

Polypeptide chain passes back and forth repeatedly across the lipid bilayer as hydrophobic alpha helices. Number and position of start and stop transfer sequences determine side of N- and C-termini

Question 34

Resident Proteins

Answer 34

ER Proteins... Some are Resident (contain ER retention signal) Some are “En Route” to other destinations.

Question 35

Soluble ER resident proteins have ____ that ___

Answer 35

PDI (protein disulfide isomerase) Catalyzes the oxidation of -SH groups on Cysteines to form disulfide bonds (S-S)

Question 36

Cytosol of the ER is a

Answer 36

reducing environment

Question 37

oligosaccharyl transferase

Answer 37

Catalyzes Protein Glycosylation Reaction Mediates the transfer of oligosaccharide to the Asn residue on the protein (**N-linked protein glycosylation**)

Question 38

Protein Glycosylation Reaction

Answer 38

Catalyzed by: oligosaccharyl transferase Mediates the transfer of oligosaccharide to the Asn residue on the protein (N-linked protein glycosylation) Enzyme is closely associated with the SEC61 complex so protein modifications occur relatively quickly following translocation into the lumen.

Question 39

Dolichol and pos charges during protein glycosylation rxn ____

Answer 39

keep sugars in place so they can be transported

Question 40

Glycan Trimming

Answer 40

Further removal and addition of sugars occurs in the Golgi apparatus

Question 41

Glycosidases

Answer 41

remove sugars

Question 42

Glycosyltransferases

Answer 42

add sugars

Question 43

Three common sugars in oligosaccharides

Answer 43

Glucose Mannose N-acetylglucosamine

Question 44

Calnexin

Answer 44

carbohydrate binding proteins ER chaperone proteins Is a lectin in er that associates with membrane, helps with efficiency of folding by sugar group (because it is a lectin)

Question 45

Precursor oligosaccharide

Answer 45

Has three terminal glucose groups that are trimmed and added to protein

Question 46

Role of glycosylation in protein folding

Answer 46

An unfolded protein (cycles of glucose trimming and glucose addition) until folded state achieved! Calexn - then glucosidase removes n terminal glucose, now N linked oligosaccharide, if properly folded, exits ER If miss folded glucosyl transferase binds - UDP glucose to UDP, glucose added on, cycle repeats.

Question 47

Retrotranslocation

Answer 47

Strict quality control Proteins that fail to achieve properly folded structure - Similar to post-translational translocation Longer time in ER - more susceptible to Mannosidase N-linked oligosaccharides as timers. Mannosidase (trimming of particular mannose), recognized by ER lectins - targets protein to the proteasome Change shape on N linked oligosaccharide (via mannosidase), recognized by retro translocation lectins - associates with translocators - associates with ubiquitin ligase - target to proteasome - protein degradation.