LEC33, 34: The Secretory Pathway & Endocytosis (Part A: Overview & Part B: The ER) Flashcards
Part A: Overview of secretory pathway and lysosomal/endosomal system Part B: The ER
what % of proteins end up in membranes or secreted from cell?
what mediates this?
>30% of proteins
secretory pathway; via the ER for protein folding and QC, Golgi for sorting proteins to diff parts of cell, and Lysosome for recycling cellular materials that can be engulfed by membrane invagination
what does transport within secretory pathway?
lipid vesicles that bud from 1 type of membrane & fuse w/ another
what are the different types of secretion that new proteins that leave golgi may be destine for? examples of each?
1) constituitive secretion: go straight to plasma membrane
2) regulated secretion: stay packages into vesicles for a bit then undergo secretion (i.e. hormone signal, neurotransmitter)
3) internal destinations: to endosome and then lysosome
these are all movements from inside cell -> outside
what is direction of protein movement in endocytosis?
outside of cell -> inside of cell
what’s the function of proteins that undergo constituitive secretion?
examples?
proteins w/ house keeping functions that’re constantly made by specialized cells, secreted all the time
albumbin, IGG, lipoproteins
collagen, fibronectin
what are examples of proteins that undergo regulated secretion?
1) peptide hormones: e.g. insulin, glucagon
2) digestive enzymes: e.g. trypsin
3) milk proteins: e.g. caesein, lactalbumin
how do we know that secretory pathway begins in the ER? describe experiment
used radioactive leucine that represented pulse in culture
at end of 3 min, chased radioactive leucine w/ cold leucine, saw as it became incorporated in different parts of cell
did x-ray imaging of cells
saw after 3 min, radioactive labeling was all in ER; after 7, in golgi; after 2 hrs, proteins in sercretory vesicles to be realsed out of cell
THUS ER = first place newly made proteins go
what % of internal cell membranes does ER constitute?
50%
what are the different types of ER / their functions?
1) smooth ER: specailized ER, contains membrane-bound enzymes important for lipid synthesis and metabolism; detoxifying enzymes esp for liver i.e. cytochrome p450s
2) rough ER: contains ribocomes, is a protein-folding compartment
why is the rough ER rough?
b/c studded w/ ribosomes
what is the nature of proteins in the ER?
proteins that’ll be secreted from the cell or exist in membranes **fold **in the ER
it is the quality control compartment for proteins
what is the difference between the outside and inside of cell’s environments? how does the ER help with this change to-come for proteins?
outside of cell: **oxidizing **environment
inside of cell (cytosol): **reducing **environment
proteins going outside cell need to be prepared for the oxidizing envinroment
thus the ER = an oxidizing environment; proteins fold in the ER within this oxidizing environment, which prepares them for the outside cell environment later
when a ribsome translates, where can protein it creates be targeted to go?
1) nucleus
2) mitochondria
3) peroxisomes
4) secreted or exist in membrane
how does a cell know where a protein will go?
protein sorting: each individual protein has **targeting sequences **that can be anywhere in the protein; these stretches say **where protein is supposed to go **
what’s the protein targeting sequence for new proteins targeted ot the ER membrane?
N-terminal signal peptide of 8-20 residues enriched in hydrophobic amino acids
often cleaved after import into the ER
what is the signal peptide? what usually happens to it?
signal peptide: on the new protein; 8-20 residues, hydrophobic a.a., often cleaved after import into the ER.
if protein has internal targeting sequence, not cleaved after import
signal peptide binds to the signal recognition particle (SRP), **ribonucleoprotein complex **that attaches to newly-synthesized proteins while they’re being translated
what happens when SRP (signal recognition particle) binds to a signal peptide? describe sequence of events
co-translational translocation
1) recognition: binding btwn signal recognition protein and new a.a. coming out of ribosome
2) arrest of translation: SRP binding to signal peptide arrests translation - physically - b/c it binds to translation center of ribosome and stops it in its tracks
3) complex of translating ribosome and SRP bind to ER membrane via SRP receptor complex
SRP receptor sits adjacent to translocation channel, the **translocon, **an **aqueous channel **where translation can resume directly into ER lumen
4) SRP dissociates b/c it binds to SRP-receptor in cell membrane; SRP dissociates; translation resumes in lumen of the ER, while signal peptide remains stuck in translocon and rest of protein loops in to ER lumen
what is the translocon?
an aqueous translocation channel
adjacent to the SRP receptor, so when SRP w/peptide binds to SRP receptor, translation resumes through the translocon channel, into the lumen of the ER
what is the nature of the translocon channel, for membrane proteins?
channel opens sideways, into plane of the membrane, and membrane-spanning domains of proteins become inserted into membrane itself
what does co-translational translocation enable for the new protein?
if want to have particle that sequesters all nascent chains of polypeptides, helpful b/c takes ribosome to membrane, and allows translation to resume in context of translocation
peptides are exposed to oxidizing ER environment this way which is crucial for proteins destined for outside of cell
what happens to signal peptide/signal sequence after translocation?
it remains embedded in the membrane of the ER b/c it was inserted directly into translocation channel
cleavage by signal peptidase
polypeptide is released into lumen of the ER, and cleaved signal peptide remains in membrane
what is the nature of the membrane-spanning portion of a membrane-bound protein?
hydrophobic, 20 a.a. long, alpha helical
how do membrane-bound proteins interact w/ translocon?
translation resumes on ER membrane until membrane spanning domain hits the translocon
then, b/c that domain is hydrophobic, it triggers a door in translocon to open, and protein swims into plane of the membrane so N and C termini each are either in cytosol or lumen of ER
what is a Type I membrane protein?
membrane protein
N terminus: in lumen
C terminus: in cytosol
what is a type II membrane protein?
N terminus: in cytosol
C terminus: in lumen
what is a complex membrane protein?
protein w/ multiple membrane-spanning domains comprising hydrohobic amino acids that form 20-25 a.a. residues
what is function of memrbane spanning domains of membrane proteins?
help either inhibit or promote translation of the loops of the protein into the lumen of the ER/the cytosol outside
act as “start transfer” and “stop transfer” sequences
what % of drugs bind to GPCRs?
50%
what is a GPI-linked anchor?
glycosylphosphatidyl inositol anchor
post-translational modification to anchor proteins to apical membrane of epithelial cells
protein is transferred to the anchor post-translationally rather than having a true membrane-spanning domain
allows much faster diffusion of membrane epithlial cells
what happens to proteins entering the ER lumen?
1) signal peptide is cleaved
2) protein gets glycosylated
3) new protein binds to ER-specific chaperones and folding enzymes
what is signal for N-linked glycosylation of a protein emerging in to the lumen of the ER?
how does this addition work?
sequence that is Asn-x-Threonine/Serine
signals addition of complex carbohydrate moiety of 14 residues
occurs via linkage to epsilon-amino group of Asn
**follwed w/ 2 residues of N-acetylglucosamine (GlcNac), 9 residues of mannose, 3 residues of glucose **
glucose residues have important roles in protein folding
why does N-linked glycosylation of some proteins entering ER lumen occur?
1) important for **protein folding **
2) **stabilizes **proteins it’s added to
3) can act as **recognition signal for when peptide is outside the cell **
4) has role in cell-cell adhesion
what aids proteins protein folding for proteins entering the ER lumen?
ER-specific form of molecular chaperones Hsp70 and Hsp90
what is calnexin? function?
specialized form of a chaperone that binds N-linked glycosylated protein in the ER while it’s folded
1) binds to final glucose unit of glycosylation complex carbohydrate moiety, keeps the protein in the ER until it’s folded
2) once folded, signal allows final glucose to be removed
3) when glucose is removed, folded protein is detached from calnexin, and then conitnues on secretory pathway
what does folding of proteins in ER depend on?
1) chaperones
2) folding enzymes
what is peptidyl-prolyl isomerase? what does it do?
enzyme that changes the configuration of proline in loop region of a protein
changes proline from cis to trans configuration, flipping protein 180 degrees, speeding up rate of folding reaction
what is protein disulphide isomerase? what does it do?
aids folding for proteins that form disulphide bonds, commonly proteins that exist outside of cell but not for intracellular proteins
proteins w/ Cysteine residues become disulfide bonds when in the ER b/c it’s an oxidizing environment
S-S- bonds thus form in the ER
but PDI works to **rearrange disulfides once they’ve arranged in the ER **so they are in the cis configuration
what is stress to the ER?
when level of unfolded protein exceeds the available # of chaperones to fold those proteins = stress; can be toxic accumulation of misfolded proteins that aggregate
what happens in type II diabetes re: ER stress?
type II diabetics’ pancrease produces massive quantity of insulin
however, insulin produced is not funcitonal
so sugar stays in blood, and signal goes back to pancreas to make mroe insulin
ER thus gets very stressed
pancreas can undergo programmed cell death b/c of stress
and level of msifolded protein in ER is so high that cells, esp insulin, completely exceeds number of chaperones available to fold the protein
how does protein deal w/ stress?
1) activation of the UPR, unfolded protein response
2) ER-associated degradation (ERAD)
what is the UPR?
signaling molecules in membrane of ER that’re very sensitive to levels of misfolded protein
when activated, results in expression of genes that encode ER-specific molecular chaperones & components of ubiquitin/proteasome pathway - so proteins can be destroyed
however proteins must be translocated back to cytosol for destruction; are not destroyed in ER
gene expression of ERAD components increase during UPR
what is ERAD? how does it work?
ER-associated degredation
proceeds after UPR
if UPR is massively unregulated, cell goes into suicide program, ERAD
luminal and membrane proteins are retrotranslocated from ER to cytosol for degradation by the proteasome in ERAD
