Membrane Traffic I Flashcards
what is the pathway that 30% of the human proteome follows?
ER –> golgi –> plasma membrane or endolysosomal system
overview of membrane trafficking pathways
- proteins are co-translationally translocated to the ER
- topology of proteins is determined at the ER- topology is always conserved across the membrane traffic
- proteins are co- and post-translationally modified
- protein trafficking requires membrane budding and fusion- requires some energy
- endocytosis leads to recycling and degradation of proteins
what are the two types of proteins that can be secreted?
- totally soluble, inside the lumen and not attached to plasma membrane and when it’s secreted, it will be out in the extracellular space
- transmembrane (anchored) proteins that are anchored to a membrane of the vesicles and delivered to the plasma membrane
discovery of secretory pathway
-looked at acinar cells and they knew digestive enzymes are secreted into the pancreatic tubes
-did EM- saw that there are vesicles called granules that accumulate in the cytoplasm and granules contain digestive enzymes
-used radioactively-labelled leucine and applied for 3 minutes to the cell and any new proteins made would incorporate this Leucine
radiolabeling of 3H-leucine to follow protein synthesis to secretion by EM
-0’ chase- right after application and you see the Leucine proteins just in the ER membrane
-17’ chase- proteins went to the Golgi exclusively
-37’ chase- they go into dense core granules and these are close to the Golgi membrane
-117’ chase- these dense core granules are close to the plasma membrane that have radioactive labelling
predictions from the membrane trafficking model
- cargoes are loaded at the ER
- proteins are required for trafficking
- cargoes are sorted and delivered to the right targets
how are the cargoes synthesized and loaded at the ER?
- signal sequence (16-30 AAs) on nascent peptides that spans the transmembrane length
- have to have signal recognition particle (SRP) and receptor on ER
- protein channel translocon (sec61) needs to be there to actually channel polypeptides into the ER lumen
what was known about protein synthesis in the ER in 1970?
-2 types of ribosomes based on EM observations
1. soluble ribosomes in the cytoplasm and not attached to any membrane (cytosolic)
2. tightly associated ribosomes with ER membrane (ER-associated)
-selective translation of specific mRNAs on these ribosomes (polysomes) and cytoplasmic ribosomes can translate the proteins that are normally in the cytoplasm
-those attached to the ER secreted protein to the extracellular space
-nascent peptides can be released into the ER
immunoglobin light chains secreted from cells are smaller than the ones made in vitro
-free ribosomes not associated with the membrane and translate it in vitro in tubes and saw that they’re running at certain size
-found that when they actually make the same protein with ER membrane the size was smaller
signal sequence hypothesis: N-terminal of the nascent peptides contain a sequence recognized for ER targeting- they are cleaved after translation
-once they’re in the ER membrane, portion gets cut
-signal sequence- part of the protein that gets cut off in the ER
signal sequence directs proteins into the secretory pathway
-hydrophobic AA residues of 10-20 AAs or 16-30- whatever spans the transmembrane
-there are usually basic residues like Arginine or Histidine that are next to the signal sequences and dictates orientation of how polypeptide goes into the ER
-cleavage site for signal peptide- usually annotated with Ala-X-Gly or Gly-X-Ala
-after ER translocation, sequences at these locations will be cut
-not all proteins have this cleavage site or basic residues but whenever it’s secreted, has to have this hydrophobic core as signal sequence
-when signal sequence appears on ribosomes, they have to be recognized by the protein –> SRP and receptor bring in complexes to the ER membrane
overview of ER translocation
-when nascent peptide comes out of ribosomes and if it has a signal sequence, this is hydrophobic and you don’t want hydrophobic things in aqueous solutions –> SRP binds to hydrophobic residues and pauses translation
-SRP then translocates onto STRP receptor on the ER membrane next to protein channel and nascent peptide goes into channel and rest of translation initiates
sec61 is IDed as the translocation channel through genetic screens in yeast
-knew signal sequences existed
-histidine in yeast is non-essential since you can make it using his4
-put his4 into ER lumen by putting a signal sequence onto this protein –> his4 only in ER lumen
-unless you supply yeast with histidine, it dies
-did random mutagenesis and supplemented yeast with histidinol and in looking for survivors of these cells, they would have mutations in his4 itself and histidine protein would be in cytoplasm
-random mutagenesis hit protein translocon called sec61 and his4 proteins stay in the cytoplasm
-mapped it to the sec61 gene and found that this is actually the protein that transforms it
sec61 sits on the ER membrane + polypeptide comes through
-typically a little plug that sits in the channel –> nothing gets translocated from the cytoplasm to the ER lumen
-as nascent peptide comes in, the plug gets translocated and the channel opens for polypeptide to come in
-this translocon has opening on one end of it and as the polypeptide comes in, the translocon opens up to the ER membrane
overview of ER translocation pt. 2
you have ribosomes with polypeptide that comes out that is hydrophobic and recognized by SRP –> SRP stops translation and SRP receptor binds SRP –> nascent peptide can go through sec61 –> rest of translation occurs in the ER
signal sequence is cleaved co-translationally
-translocon acts as pacman- as signal sequence comes in, opens up laterally and escapes from this
-there’s a signal peptidase that recognizes particular AA sequence and cuts it
-signal sequence stays in the ER membrane
-signal peptide peptidase cuts the sequence off and it disassociates from ER membrane and can be broken down into amino acids
-protein assembled and folded inside the ER lumen and not attached to the membrane anymore –> soluble protein and secreted out of the cell
-signal sequence is cleaved co-translationally and as the protein is translated and signal sequence is made, gets cut
transmembrane and membrane-anchored proteins
- have additional hydrophobic patches on polypeptides
- have a c-terminal hydrophobic patch (with or without signal sequence)
exception: post-translational translocation
how do you generate a transmembrane protein?
when there are two hydrophobic patches, one gets to the ER then signal peptidase cuts it and the rest of translation keeps going in the ER lumen then a second hydrophobic patch comes into the translocon but with no cleavage sequence, it gets stuck in the ER membrane and translation occurs in the cytoplasm
topology is determined during biogenesis in the ER
-type I- N-terminus in the ER lumen with cytoplasmic tail
-type II- N-terminus in the cytoplasm and C-terminus in lumen of the ER
-type III- a little bit of the N-terminus on the ER and most of the C-terminus in the cytoplasm
-type IV- multi-pass- multiple transmembrane domains
-these are all dictated by the AA sequence
signal sequence is not always at the N-terminus
-N-terminus but signal sequence wasn’t at the tip of the N-terminus
-most of the N-terminus is made in the cytoplasm by ribosomes and when the signal sequence hits, recognized by SRP and translocates into translocon on the ER membrane
-if you have an amino acid charge (basic net charge), positive charge stays in the cytoplasm
-if you have Arginine, Histidine, and Lysine, these will stay in the cytoplasm
-orientation determines whether you have these basic residues or not
polytopic membrane proteins: multiple start and stop sequences
you can have multi-pass on transmembrane proteins if you have more stop transfer sequences or signal sequences
hydropathy plots can help predict topology
you can determine how many transmembrane domains this particular protein may have based on hydrophobic residues
what are other approaches to determine membrane protein toplogy?
-make antibodies against certain locations on proteins and see if that’s accessible from extracellular space (accessibility of antibody epitopes)
-add epitopes like his-tag or alpha-tag onto a protein in known locations and you can probe for that using antibody and see if it’s accessible from extracellular space
-add additional glycosylation sites into proteins and this changes the size of the proteins when you do westerns
what is another way to make transmembrane proteins?
-C-terminal hydrophobic patch
-you have a signal sequence that’s cleaved and make most of the proteins in the ER lumen
-as it was completing translation, last bit of the protein has hydrophobic patch
-instead of using amino acid to anchor the proteins onto the membrane, lipid anchors it to the membrane
-you have GPI-anchored proteins through the secretory pathway
without signal sequence: tail-anchored proteins are inserted into the ER membrane through the GET pathway
-all of the proteins are generated in the cytoplasm and no signal sequence in protein –> protein translation occurs in cytoplasm on the ribosome
-as translation was finishing, there was a hydrophobic patch on the protein –> recognized by pre-targeting complex and brings proteins onto the GET pathway proteins on the ER membrane and gets inserted with ATP
-proteins get anchored to the ER membrane after insertion and go through the secretory pathway
-SNARE proteins- synataxin and synaptobrevin use this pathway to be inserted into the ER
what are the two types of cargoes that can be loaded in the ER?
- secreted (soluble) proteins with no transmembrane domain
- transmembrane proteins that can be made if you have a signal sequence that cannot be cleaved or if you have multiple hydrophobic patches on the membrane
protein folding in the ER
- chaperones help with folding the proteins inside the ER lumen
- sugar modification on the proteins that are secreted
- disulfide bonds that can be made on assisting residues on the proteins –> ER is oxidizing environment
folding begins co-translationally and involves chaperones
-chaperones like BIP- if there’s hydrophobic residues on amino acids, these try to fold it so the residues aren’t exposed on the surface of proteins
-glycosylation happens on the Arginine residues on a protein- they have a consensus sequence and if you have Asparagine or any amino acid they get glycosylated
-important for hydration of the cell surface since they attract water molecules to the surface and important for antibody specificity
disulfide bond also forms in the ER since there’s an oxidizing environment
done by protein disulfide isomers (DPI)- make sure disulfide bond is made correctly
what happens if a protein doesn’t fold properly?
if protein folding doesn’t happen properly, there’s a process called E-rad that translocates the protein and sends it back to the cytoplasm for degradation
topology video
-mRNA in the cytoplasm and free ribosome locks on to begin translation
-tRNA cycles through ribosome, matching codons defined by mRNA and attaches amino acid
-first signal sequence exits the ribosome- signal recognition particles (SRPs) bind the signal sequence and block the ribosome from translating more
-SRP ribosome complex will diffuse to the ER membrane due to signal sequence and SRP alerting the cell that this protein should be created in the ER
-SRP attaches to the SRP receptor and the protein sequence is inserted into translocon
-side of signal sequence with more positively charged amino acids is kept in the cytoplasm
-protein can be inserted head or elbow first to keep the positive side in the cytoplasm
-ribosome creates seal with translocon and SRP detaches
-as the protein was inserted head first, ribosome creates the protein in the cytoplasm
-N-glycosylation domain is recognized by proteins in the ER and oligosaccharide is attached
-translocon will likely open a port to eject the hydrophobic domain of the protein into the ER membrane
-second signal sequence is created and SRP detects it and holds translation
-ribosome gets diffused again to ER but this time the more positively charged side is flipped –> goes elbow first
-any unlinked glycosylation site will not be used unless they’re in the ER lumen
-third hydrophobic domain enters the translocon with positive amino acid towards cytoplasm and ribosome detaches
