Protein Sorting

continuous with the outer membrane of the nuclear envelope consists of vast networks of tubules which extend throughout cytoplasm organelles arent completely discrete RER- responsible for protein synthesis SER- responsible for lipid synthesis eg phospholipids

synthesis of lipids eg phospholipids +cholesterol- forming cell membranes synthesis of steroid hormones eg testosterone + cortisol storage and release of Ca2+ from sarco retic detoxification eg of alcohol- lots of SER in liver

site of protein synthesis and processing for mebrane bound proteins destined for ER, golgi, plasma membrane, lysosomes, endosomes transmembrane and to be secreted from the cell

in cytosol small and subunit bought together in ribosome section of mrna- coding for specific protein- is bound to ribosome protein is transcribed and leaves ribosome

same as above but protein binds to ER and transferring the protein into the cell from the outside

when proteins are imported into the ER during protein synthesis one end of the protein attaches to ribosome, other end inserts itself to ER, protein will remain in primary sequence once a protein has been translated and entered ER it will be modified- fold into 3D conformation (ionic,H, disuplhide bonds + van der waals) and glycosylation

• recognition signal recognition particle (SRP) will recognise signal on N terminal of growing polypeptide binds to signal sequence and ribosome - (pauses synthesis- bring about translational pause domain) SRP consists of RNA and 6 proteins 2.targeting signal sequence must be recognised by ER and embedded within ER membrane SRP receptor on RER membrane brings it close to protein translocator, protein moves through translocator and enter ER 3.release once ribosome has translated, mRNA is released back to free ribosome pool SRP released from receptor and is recycled mrna may still remain bound to ER as it can be translated to multiple ribosomes

if a protein is to be released fro cell, it will be fully transported into ER lumen if a protein is destined to be a membrane protein, it will get embedded into the ER membrane

addition of a sugar to a protein very specific to proteins that enter the ER- quality control, recognition, protection precursor oligosaccharide composed of 14 sugars is added to proteins via N terminus of asparagine side chain (N linked glycosylation) occurs during protein synthesis as proteins enter ER lumen

quality control- recognises correctly folded proteins 1. if protein is not folded correctly 3 glucose and 1 mannose are cleaved from N linked oligosaccharide 2. still not correct, glucosyl transferase enzymes add single glucose back (proteostasis) (signal to calnexin) 3. calnexin binds to unfolded protein to prevent aggregation 4. removal of terminal glucose releases protein from calnexin 5. glucosyl transferase determines if protein is correctly folded, if not a single glucose is added back if protein remains misfolded, it will be released from ER for targeted degradation within cell

misfolded protein accumalate in the cell, causes ER stress and trigger UPR -inhibits protein synthesis -degrades misfolded proteins -increases transcription of chaperone proteins incl BiP, calnexin apoptosis (cell death) may occur if problem persists

used to determine how things can move around a cell 1.addition of radiolabelled amino acids to cell 2-newly formed proteins are radiolabelled (pulse) washed away radiolabelled amino acids (chase) 3. cells returned to non radioactive medium (chase) 7 min- into golgi 120 min- into vesicles which leave the cell

for vesicular cargo to reach target organelle, membranes must fuse (using SNARE proteins) V-SNAREs (vesicle) and T- SNARE(target)- specific to one another wrap around each other to form stable trans-SNARE complex draw vesicle closer and closer until it can fuse releases cargo inside membrane during fusion, a Rab-GAP (GTPase activating protein) causes Rab to hydrolyse GTP to GDP Rab-GDP is then released from the vesicle and bound by Rab-GTP dissociation inhibitor (GDI) to keep Rab inactive (recycled)

COPII coated vesicles bud off from ER carrying cargo (protein) mutiple vesicles can fuse (SNAREs must match) forming vesicle tubular clusters (pathway from golgi back to ER= retrieval pathway)

stacks of flattened membrane enclosed compartments called cisternae cis and trans face cis golgi network receives things from ER trans GN secretes onwards from ER unclear how things move through golgi- by vesicles/ network is fluid

further processing of oligosaccharide chains -ensures correct folding of proteins -prevents unwanted aggregation -acts as a signal for sorting and targeting correct pathway

function to digest unwanted material contain many enzymes to break down macromolecules acid hydrolases active at 4.5-5pH - very acidic + enzymes only active at this pH degradation occurs inside lysosome pH maintained via vacuolar ATPase tagged with mannose 6 phosphate

types of lysosomes transporters of material that has been digested by the cell (endocytosis) fusion of late endosomes with endosomes of lysosomes generate endolysosomes- become more acidic once digestion of endocytosed material is complete, they form lysosomes

acid hydrolases (in lysosomes) tagged with mannose 6 phosphate mp6 directs proteins to lysosomal network mannose binds with hydrolase precursor (want to get into lysosome) P=GLcNAc added which adds on phosphate tag (M6P signal) + moves through golgi network binds to M6P receptor on edge of golgi clathrin coated endosome containing hydrolase transported to early endosome (will become lysosyme) fuses with membrane vesicle is recycled/ retrieved

transport into cell from plasma membrane after endocytic vesicle fuses with early endosome, ingested material can be either degraded- moves towards cell and membrane composition alters, no longer recycles- starts degradation recycles- vesicles containing unwanted material for recycling to plasma membrane bud off and fuse with recycling endosome

two types of pathway -consitutive secretory pathway- all of the cargo is released at once -regulated secretory pathway- cargo is maintained in vesicle until its needed relies on signal such as hormone or neurotransmitter eg digestive cells (enzymes after eating), endocrine cells (insulin)

clatharin- from plasma membrane (endocytosis) + between golgi and endosomes cop1 coated- from golgi cop2 coated- budding from ER

Rab GTP tells vesicles which membranes are correct to bind to GTP bound- active GDP bound- inactive GEF found on vesicle membrane activates Rab- activates Rab-GDP in cytosol to form Rab- GTP Rab GTP binds to Rab effector protein on target membrane- tethering of vesicle to target membrane