protein modification and quality control Flashcards
what are the functions of the endoplasmic reticulum?
- insertion of proteins into membranes
- formation of S-S bonds
- proper folding of proteins
- glycosylation
- specific proteolytic cleavage
- assembly of multi-subunit proteins
where and how do disulphide bonds form?
occurs specifically in the lumen of the endoplasmic reticulum. disulphide bonds are formed when two cysteine residues become oxidised to form a covalent bond
which enzyme catalyses disulphide bond formation in the endoplasmic reticulum?
protein disulphide isomerase
what are the functions of PDI?
can allow disulphide bonds to form in the target protein
can rearrange disulphide bonds into the correct conformation
what is the purpose of pepetidyl-prolyl isomerases?
accelerate the interconversion of cis and trans isomers of proline residues
what is N-linked glycosylation?
addition of oligosaccharides to the amino group of asparagine residues. occurs in the endoplasmic reticulum
what is the oligosaccharide that is added to the polypeptide in N-linked glycosylation?
two N-acetylglucosamines
nine mannose sugars
three glucose sugars
outline the process of assembly of the oligosaccharide for N-linked glycosylation
1- phosphate of UDP is added to the dolichol with N-acetylglucosamine
2- sugar residues are added individyally
3- five mannose sugars are delivered as GDP-mannose sugars
4- when there are five mannose sugars exposed to the cytoplasmic face, the dolichol is flipped into the membrane
5- subsequent mannose sugars are delivered one at a time, carried by dolichol phosphate
how are precursor oligosaccharides transferred to proteins in N-linked glycosylation?
oligosaccharide transferase
what is the importance of glucose trimming after N-linked glycosylation?
quality control mechanism
-unless the oligo is correctly trimmed, the protein will be trapped in the endoplasmic reticulum and will not move to the Golgi
what is the purpose of chaperone proteins?
- assist protein translocation
- retain unfolded proteins in the ER
- mediate the unfolded protein response
what is BIP?
binding immunoglobulin protein
binds to exposed amino acid sequences that would normally be buried on the exterior of a folded protein
what is calnexin?
an ER membrane associated protein that binds to oligosaccharides on incompletely folded proteins
what is calreticulin?
a soluble ER protein that binds to oligosaccharides on incorrectly folded proteins
outline how calnexin/calreticulin mediate quality control in the ER
1- unfolded protein has glucose sugars removed by a glucosidase
2- when the protein has one glucose sugar left, it triggers binding of calnexin or calreticulin
3- once bound to calnexin, a glucosidase moves the last glucose
4- if the protein is correctly folded, it exits the ER and passes to cis-golgi
5- if not correctly folded, glucosyl-transferase adds a sugar, causing it to bind calnexin again
what happens if there are problems with protein folding?
- proteins might be trapped in the wrong conformation
- proteins may be incorrectly associated with other subunits
what is the unfolded protein response?
a process which allows the cell to produce more chaperones and unfolding catalysts in response to high levels of unfolded protein
explain how BIP and Ire1 respond to unfolded protein
BIP is normally bound to Ire1 to keep it in the inactive
form
when there is high levels of unfolded protein, BIP dissociates
this allows Ire1 to dimerise, activating it and allowing it to exert endonuclease activity on mRNA, this produces a mature mRNA and acts as a transcription factor to produce more chaperones
what does PERK do?
it is a kinase resident in the ER membrane that dimerises in stressed cells to phosphorylate eIF2, inactivating it and reducing protein synthesis
what happens to misfolded proteins that cannot be refolded?
they are bound by chaperone proteins and translocated out of the endoplasmic reticulum
what is ubiquitin?
a 76 amino acid protein that attaches to lysine. polyubiquitination targets proteins for proteasome-mediated degradation
what is the Golgi?
membrane bound sacs known as cisternae. each cisterna carries Golgi enzymes which help modify cargo proteins
what are the five functional regions of the Golgi apparatus?
cis Golgi network, cis-golgi, medial Golgi, trans-golgi, trans-golgi network
