HC14 glycosylation and vesicular transport Flashcards
Signal sequence for protein transport into ER
N-terminal signal sequence: hydrophobic residues, followed by some polar residues.
Transport into ER: co-translational translocation
Signal recognition particle (SRP) recognises signal peptide –> bind with low Km to signal –> further synthesis of protein is stalles –> SRP receptor on ER recognises ribosome-SRP-protein complex –> receptor interacts with translocator protein ( plug is replaced) –> translation proceeds again –> translocation begins –> signal sequence is cleaved –> free soluble mature polyptided chain in ER lumen
These translocator protein channels are hermetically sealed from cytosol: cytosolic I- cannot bind to ribosomes bound to ER membrane, but can bind to free cytosolic ribosomes.
ER transmembrane proteins: signal sequences + N/C-terminal on which side of membrane
1: start transfer sequence–> gets spliced off –> internal stop transfer sequence
2: internal charged sequence: + charged = C-terminal: outside of ER membrane: - charged = N-terminal: inside ER lumen.
If C-terminus was to be inside ER lumen and N-terminus outside: flipped internal charged start-transfer signal: + side on N-terminal.
both termina outside on ER membrane: double-pass: start-transfer and stop-transfer.
Glycosylation: types
O-glycosylation: on OH-group of serine/threonine
N-glycosylation: asparagine group. Placed on in the ER lumen. Modifications of glycosylation in Golgi.
Glycosylation in the ER: process
dolichol gets phosphorylated on cytosolic membrane –> sugar molecules get coupled to dolichol (energy from UDP,CDP,GDP that carrier the molecules previously) –> eventually chain flips to ER lumen.
Other dolichol molecules bind 1 sugar –> flip to inside –> transfer the sugar molecules to the growing sugar tree.
the whole sugar tree gets transfered to the protein
Quality control for protein folding in ER by?
Protein glycosylation. ER proteins have a sugar tree with 3 glucose molecules attached to it –> 2 will get trimmed of (glucose trimming) –> calnexin helps fold the protein –> folding correct? –> glucosidase cuts of last glucose –> protein can exit ER.
folding incorrect? –> glucosyl transferase recognises this –> glucose group is put back –> again calnexin –> correct folding? –> etc.
a lot of misfolded proteins:
misfolded proteins can bind to transmembrane kinases –> dimerisation and phosphorylation of kinas –> endoribonuclease activity –> splicing of pre-mRNA –> mRNA –> translation –> transcription regulator protein is synthesised –> enters nucleus –> activation of genes encoding for chaperone proteins –> chaperones mRNA –> cotranslation translocation into ER –> help protein folding –> more chaperones –> higher Vmax
Vesicular transport: coats
COP1: Golgi coats
COP2: ER coats
Clathrin coating enables vesicle formation. cargo proteins bind to cargo receptors –> dimerisation of receptor –> clathrin can bind adaptor proteins on cytosolic side of membrane –> dynamin –> GTP hydrolysis –> brings membranes close together –> membrane fusion –> results in budding of the vesicle from membrane –> coating is released –> naked vesicle
Fusion of membranes: vesicle fusion
Rab and SNARE proteins.
v-SNAREs on naked vesicle, have their own Rab protein –> interact with t(arget)-SNAREs. Rab proteins associate with tSNAREs. –> v-SNAREs bind to t-SNAREs –> membrane fusion –> Rab-GTP gets hydrolysed –> Rab-GDP
How to reatin proteins into the ER?
Interactions with COP1 (golgi specific coat) and KDEL sequence (lysine, aspartic acids, glutamic acid, leucine)
Endo-H, Endo-F
endoglycidases. Endo-H can cut of sugars before the trans-golgi. Endo-F cuts all N-glycosylated sugars.
How to target enzymes to the lysosomes?
via Mannose-6-phosphate.
in ER: lysosomal enzymes are decorated with M6P by GlcNAc phosphotransferase –> in trans-golgi: M6P binds with high affinity to M6P receptor –> in lysosome: low pH –> M6P becomes protonated –> Km of M6Preceptor binding rises –> protein lets go of receptor –> delivery of lysosomal enzyme into lysosome.
Recycling of receptors by?
Lysosomes. for instance LDL receptor.
GLUT4 glucose transporter
Synthesised by muscle and adipose tissue. Store the receptor in secretary vesicles.
When cells are stimulated with insulin –> GLUT4 relocalised to plasma membrane –> more enzymes –> Vmax of glucose uptake increases, Km stays the same!
