lecture 4 Flashcards
where do cytosolic ribosome-made proteins end up
- remain in cytosol
- or are TARGETED to intracellular organelles
- e..g ER, mitochondria, chloroplasts, peroxisomes, nucleus
** must have specific signal sequence
where do ER ribosome-made proteins end up
- SORTED to PM, Golgi, lysosomes or can remain in ER
what are the two major protein-sorting pathways
consecretory and secretory
nonsecretory pathway
- proteins that go to lysosome
secretory pathway
- proteins that go to secretory vesicle
ER
- uninterrupted membranous tubules and vesicles separated from cytoplasm
- RER has ribosomes on the tubules
what are the cisterna
- sacs/tubules that make up the ER?, inner parts of the tubules
where does the eR extend from
nucleus
what does the TEM allow us to see, that light microscopy would not
- the ribosomes
in what direction do ER ribosomes face
- towards the cytosol
what did experiment 1 with the detergent show?
- that secreted proteins must first go through the ER
describe the experiment that showed that secreted proteins must first go through the ER
- we know that antibodies are examples of proteins that are secreted outside of the cell
- so they homogenized cells that expressed a certain antibody
- homogenization disrupts the ER, and breaks it apart
- ER re-forms as vesicles called microsomes
- microsome splits into two
- 1/2 is treated with non ionic detergent first, and then protease is added to chew up the immunoglobin
- 1/2 is treated only with protease
- after running SDS page, second example still has protein present
- adding detergent allowed the protease to get in and chew up the immunoglobulin, otherwise cannot passed microsomal membrane.
how do we know that translocation and translation occur simultaneously?
- experiment involving in vitro translation
1) all the pieces of the translation apparatus were put in a tube with a specific mRNA. allow the protein to be synthesized for half an hour, and then add the microsomes. when these proteins were analyzed by SDS page, it was observed that none of the protein was found in the lumen of the microsomes.
2) mixing everything together, mRNA, in vitro translation concoction, microsomes. secreted protein was now found in the lumen of the microsome, and had undergone furtherr processing to cleave off a certain piece
** this shows that translation and translocation occur simultaneously
translocation of proteins
- ## movement of protein from outside into lumen of ER
nascent proteins
- newly initiated polypeptide
where is signal sequence located
amino terminal?
where is the SRP receptor embedded
ER membrane
SR[
signal recognition particle
translocon
protein channel
cleavage site
- where signal sequence is cut by a signal peptidase
what protein modifications take place in the ER
- specific proteolytic cleavage (signal peptidase cleaving off the signal sequence)
- glycosylation (addition of sugar groups)
- formation of covalent disulfied bonds
- folding of polypeptide chains with the help of helper proteins
glycosylation in the ER
- there is a complex sugar structure made up of 14 residues
- it is attached to the dolichol on the membrane, linking the sugar structure to the membrane
- proteins that start to emerge in the lumen of the ER will be recognized by portions called oligosaccharyl transferases, which will transfer the oligosaccharide onto an asparginine residue of the polypeptide.
- the glycosides can work to modify N glycoln which is covalently attached to the protein at the Asp residues.
what is the purpose of glycosylation
- it has functions in protein folding, conferring protein stability, cell adhesion, e.g. EC matrix proteins are sticky and glycosylation and sugar residues are responsible for this stickiness
what proteins contribute to proper folding of proteins in the ER (after glycosylatio> )
BiP, lectins calnexin and calreticulin, and PDI
- they present the midsolfing or aggregation of nascent proteins
when can proteins be transported fom RER to GOlgi
only when they are properly folded
why are helper proteins important for protein folding
- cotranslation into the ER lumen is that it can make the cell vulnerable to misfiolding or aggregation
- hydrophobic amino acids will try to stick to each other, so chaptern proteins will prevent them from misfolding
BiP
- ER chaperon prtotein that binds directly to emerging polypeptide that may start to misfold, usually on hydrophobic stretches.
oligosaccharyl transferase function for protein folding in the ER
- might receive a signal to stick on a branched sugar to maybe a hydrophobic stretch of amino acids or part of the peptide that is starting to misfiled.
- this can allow it to be recognized by other chaperon proteins (calnexina dn calreticulin)
where is calretifuclin found
- floating in the eR
where is calnexin found
- in the ER membrane
PDI (another helper protein)
- forms transient disulphide bonds to hold on tot eh sulfur atoms to help form and properly form those sulfur bonds for structural stability.
targeting
directs proteins to the right destination (organelles), happens during or after synthesis
sorting
directs proteins to the lysosomal and secretory pathways (ER —> Golgi, lysosomes)
stringing the concepts together
- identify cellular features by microscopy isolate and homogenize to free the organelles
- use sucrose desinity-gradient centrifugation of homogenate to allow for isolation of microsomes and ribosoomes
- use SDS page to identify newly translated proteins
what is the difference between the two types of protein synthesis (microsomes present vs not present)
- when the microsomes are present, it results in the creation of a mature protein that has a signal sequence
- when the microsomes are not present, there is not incorporation into microsomes, and therefore no removal of signal sequences
