Ch 12 Pt 2 Flashcards
The ER is organized into a
netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol
The ER and nuclear membranes form a
continuous sheet enclosing a single internal space, ER lumen
SER is the primary site of
ER vesicle budding, transitional zones.
In specialized cells the SER is involved in
lipid metabolism, and steroid biosynthesis.
Site of detoxification (ex. liver has lots of smooth ER) Sarcoplasmic reticulum - smooth ER ca2+ storage center
SER has a network of
Network of tubules
RER is the primary site of
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
RER is associated with
ribosomes
ER breaks into fragments, fragments reseal to form closed vesicles called
microsomes: small version of ER, retain functionality of ER
can study as if is really the ER
Smooth vs Rough microsomes
Rough - more dense - further down.
can use a density (ex. sugar) at right density float vs sink
G. Blobel and Dobberstein, 1975 experiment did what
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.
Blobel’s experiment showed
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
if, in Blobel’s experiment, it was a soluble cytosolic protein being looked at what would the results be
it would be the same size on the SDS page because it is not transported into the ER for cleavage
Are secreted proteins immediately secreted out of the cell after ribosomes synthesize them in the cytosol
No, They go to the ER
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?
No, because the protein is inside of the rough ER (microsome) where the enzyme cannot get to it
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?
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
Co translational import
translation occurs while translocated
signal sequence associates with binding site
ribosomes associate with er membrane
signal peptidase, associated with translocator, cleaves as being translated
Two types of proteins made in the ER
Membrane associated
soluble
ER Signal Sequence Directs ____ to ER membrane
Has ___ at its center
Binds to ___
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
SRP
in ___
___ lined with ____
(Signal Recognition Particle)
rodlike ribonucleoprotein
(six polypeptides and one RNA)
in cytosol
Large hydrophobic pocket lined with Methionines
SRP wraps around
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
SRP binds to ER sequence and SRP receptor steps
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
Free VS membrane bound polyribosomes
Ribosome is not fixed to the ER
Sec 61 is a ___ that forms ___
It is the ___
___ gating
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)
Steps of the Translocation of a Soluble (Lumenal- Secreted) Protein Across the ER
- ER signal sequence binds to a specific binding site in the pore, serves as a start-transfer signal that opens the pore
- Signal sequence remains bound to Sec 61 complex during translocation
- Signal sequence also in contact with lateral seam (lateral gating)
- Signal peptidase cleaves signal sequence, mature protein released into lumen.
Single Pass TM protein
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
Single Pass TM protein when N terminus-Cytosol
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.
If pos charged AA follow the start transfer sequence
the orientation is the opposite (N- terminal-lumen side)
Multipass TM protein
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
Soluble ER resident proteins have ____ that ___
PDI (protein disulfide isomerase)
Catalyzes the oxidation of -SH groups on Cysteines to form disulfide bonds (S-S)
Protein Glycosylation Reaction
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.
Dolichol and pos charges during protein glycosylation rxn ____
keep sugars in place so they can be transported
Glycan Trimming
Further removal and addition of sugars occurs in the Golgi apparatus
Three common sugars in oligosaccharides
Glucose
Mannose
N-acetylglucosamine
Calnexin
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)
Role of glycosylation in protein folding
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.
