CBIO 3400 Week 6 Study Questions 1 What is the KDEL sequence How does it affect the sub cellular localization of proteins Describe the complete mechanism regarding the KDEL sequence and protein trafficking KDEL is a sorting signal sequence attached to the KDEL receptor protein in the membrane of the Golgi this KDEL receptor grabs soluble ER resident proteins from the Golgi The low acidic pH of the Golgi allows the KDEL receptor to attach to the ER resident proteins within the Golgi When secretory vesicles break off of the Golgi the KDEL receptors go with the vesicles still holding on to the ER resident protein it grabbed in the Golgi When the secretory vesicles meet and fuse with the ER membrane the high basic pH of the ER causes the KDEL receptor protein to release the missorted ER resident protein into the lumen of the ER In general the KDEL sequence affects the sub cellular localization of proteins by bringing them back to the ER from the Golgi The complete mechanism regarding the KDEL sequence and protein trafficking is as follows DATA CBIO3400 1 A vesicle containing an ER resident protein with KDEL The cid 3 KDEL cid 3 receptor peptide attached begins to form from the membrane of the rough ER The membrane of the soon to be KDEL cid 3 cid 882 The cid 3 ER cid 3 retention cid 3 signal vesicle contains a KDEL receptor and is surrounded by KDEL cid 882 receptors cid 3 recognize cid 3 and retrieve cid 3 proteins cid 3 from cid 3 Golgi a COPII coat The ER resident protein is NOT associated with the KDEL receptor at this time 2 The vesicle buds off of the rough ER membrane and K Lysine D Aspartic cid 3 acid E Glutamic cid 3 acid L Leucine heads toward the Golgi 3 The vesicle fuses with the Golgi releasing the missorted ER resident protein with KDEL peptide attached into the Golgi lumen and the KDEL receptor into the Golgi membrane 4 The missorted ER resident protein with KDEL peptide attached binds to the KDEL receptor and a vesicle begins to form from the membrane of the Golgi The membrane of the soon to be vesicle contains the KDEL receptor with its attached missorted ER resident protein and is surrounded by a COPI coat To reiterate the ER resident protein IS associated with the KDEL receptor at this time 5 The vesicle buds off of the Golgi and heads toward the rough ER 6 The vesicle fuses with the rough ER releasing the ER resident protein with KDEL peptide attached back into the ER lumen and the KDEL receptor back into the rough ER membrane 1 2 How do proteins migrate through the Golgi Compare and contrast two existing models There are 2 existing models regarding how proteins migrate through the Golgi 1 the vesicular transport model and 2 the cisternal maturation model In the vesicular transport model proteins are brought from the ER to the Golgi in vesicles that fuse to the CGN these vesicles then bud off and fuse again with each cisternae in the Golgi until they finally bud off of the TGN and head toward either the plasma membrane or the endosome In the cisternal maturation model proteins are also brought from the ER to the Golgi in vesicles that fuse to the CGN however the proteins are then transported directly through the membranes of the cisternae NO USE OF VESICLES until they reach the TGN Upon arriving at the TGN the proteins bud off in vesicles and head CBIO3400 toward either the plasma membrane or the endosome Large Golgi products pass through the cisternae in this manner via cisternal maturation progression How cid 3 are cid 3 proteins cid 3 migrating cid 3 through cid 3 the cid 3 Golgi CBIO3400 DATA cis trans DATA Mannose cid 882 6 cid 882 phosphate added cid 3 in cid 3 the cid 3 Golgi cid 3 is cid 3 the cid 3 sorting cid 3 signal Two cid 882 step cid 3 process Cis cid 3 GlcNAc cid 882 phospho cid 882 transferase 2a cid 3 2b cid 3 2g 3 Describe how lysosomal hydrolases are transported from the Golgi to the lysosome M6P cid 3 receptor cid 3 cycles cid 3 between Golgi cid 3 and cid 3 early cid 3 endosome Lysosomal Trans cid 3 uncovering cid 3 enzyme cid 3 hydrolases moves NAGPA cid 3 N cid 882 acetylglucosamine cid 882 from the ER 1 cid 882 phosphodiester cid 3 alpha cid 882 N cid 882 vesicles cis Golgi acetylglucosaminidase cid 3 trans Golgi vesicles lysosomes Lysosomal proteins have a complex signal patch Mannose 6 PO4 M6P acts as the sorting signal M6P receptors bind M6P modified proteins and coat adaptors with a clathrin coat GlcNAc phosphotransferase adds GlcNAc PO4 to mannose in the CGN GlcNAc is removed in the TGN leaving only mannose PO4 behind M6P receptors can then bind to lysosomal proteins in the TGN where the pH is 6 7 but must release their cargo in the late 2 endosome where the pH is 6 0 hence M6P receptor binding is pH dependent The removal of from the cargo and the low pH in the lysosome make transport unidirectional After the the PO4 is removed from M6P it becomes mannse 6 which cannot bind the M6P receptor M6P PO4 receptors are thus recycled from the early endosome back to the TGN using a retromer coat CBIO3400 4 Compare and contrast three different kinds of endocytosis PINO CYTOSIS fluids small vesicles involved budding off and clathrin coated vesicles solids phagosomes pseudopodia growth around a PHAGOCYTOSIS particle is driven by actin polymerization occurs with all eukaryotic cells recycling of plasma membrane clathrin coated pits clathrin coated vesicles early endosomes specific example caveolae caveolae require other a triggered process antibodies are best known trigger particle binds to cell membrane via receptors signal transmitted to cell Rho GEF activation Rho proteins to pinch off vesicle use dynamin instead of clathrin coat to pinch off DATA vesicle transcytosis Receptor cid 882 mediated cid 3 Endocytosis may fuse to endosome or not GTPase actin polymerization PI 3K The cid 3 LDL cid 3 receptor RECEPTOR MEDIATED ENDOCYTOSIS macromolecules bind to specific transmembrane receptors receptors cluster in clathrin coated pits some always associate with clathrin pits while some only associate with ligand cargo 1 000 receptors pit mixed receptor population endocytosis and fusion to early endosome 3 different fates 1 recycling e g LDL receptor transferrin receptor CBIO3400 2 transcytosis e g Fc receptor binds to antibodies and 3 degradation e g signaling hormone receptors like opioid receptors receptors retrieved from the endosome and sent back to the Golgi are recycled transcytosis is when receptors move to a different part of the plasma