Quiz 2 Material Endomembrane System ER Protein Import ER Protein Folding Intracellular Vesicle Transport Golgi Endocytosis Lysosomes Exocytosis Golgi KDEL Pathway slide 1 of 2 DATA CBIO3400 The cid 3 KDEL cid 3 receptor KDEL sorBng signal sequence of AAs lysine asparBc acid glutamic KDEL cid 3 cid 882 The cid 3 ER cid 3 retention cid 3 signal KDEL cid 882 receptors cid 3 recognize cid 3 and retrieve cid 3 proteins cid 3 from cid 3 Golgi pH gets more more acidic acid leucine from ER Golgi K Lysine lysosome D Aspartic cid 3 acid helps control protein E Glutamic cid 3 acid sequence interacBons L Leucine higher more basic pH causes KDEL to release protein in ER lower more acidic pH causes KDEL to grab protein in Golgi DATA CBIO3400 KDEL Pathway slide 2 of 2 DATA CBIO3400 The cid 3 Golgi cid 3 a cid 3 stack cid 3 of cid 3 Pita cid 3 breads Golgi cid 882 The cid 3 major cid 3 sorting cid 3 center cid 3 for cid 3 membrane cid 3 and cid 3 secreted cid 3 proteins cid 882 Cisternae cid 3 4 cid 882 6 cid 3 or cid 3 more cid 882 Addition cid 3 of cid 3 O cid 882 linked cid 3 oligo cid 882 sacharides cid 882 Modification cid 3 of cid 3 N cid 882 linked cid 3 sugars cid 882 Resident cid 3 proteins cid 3 are cid 3 linked cid 3 to cid 3 membrane transmembrane glycosidases and cid 3 glycosyl transferases major sorBng center for membrane secreted proteins cisternae 4E6 or more addiBon of OQlinked oligosaccharides modi caBon of NElinked sugars resident proteins linked to membrane TM glycosidases transferases N cid 882 acetylneuraminic acid cid 3 NANA NQLinked Oligosaccharide Processing in the Golgi can serve as sorBng DATA CBIO3400 signals 2 main types of NElinked N cid 882 linked cid 3 oligosaccharide cid 3 processing cid 3 in cid 3 the cid 3 Golgi cid 882 Can cid 3 serve cid 3 as cid 3 sorting cid 3 signals cid 882 Two cid 3 main cid 3 types cid 3 of cid 3 N cid 882 linked cid 3 sugars cid 3 complex cid 3 and cid 3 high cid 882 mannose cid 882 Many cid 3 variants cid 3 hundreds cid 3 of cid 3 Golgi cid 3 glycosyl transferases in cid 3 human cid 3 genome sugars complex endo HEresistant highEmannose endo HEsensiBve will become sorBng signal to lysosome many variants will cid 3 become cid 3 sorting cid 3 signal cid 3 to cid 3 the cid 3 lysosome DATA CBIO3400 cis Vesicular Transport Model How cid 3 are cid 3 proteins cid 3 migrating cid 3 through cid 3 the cid 3 Golgi trans cisternae stay in place vesicles bud o each cisternae fuse w next cisternae each cisternae keeps its enzymes i e enzymes don t have to be transported back b c cisternae aren t moving DATA CBIO3400 cis How cid 3 are cid 3 proteins cid 3 migrating cid 3 through cid 3 the cid 3 Golgi Cisternal Matura2on Model trans cisternae mature move toward TGN necessary enzymes must be transported back to less mature cisternae via vesicle transport large Golgi products pass through via this model were not observed in vesicles What is the KDEL sequence How does it a ect the subQcellular localiza2on of proteins Describe the complete mechanism regarding the KDEL sequence protein tra cking KDEL is a sorBng sequence aJached to KDEL receptor protein in KDEL a ects subEcellular localizaBon of proteins by bringing them Golgi membrane back to ER from Golgi 1 Vesicles COP2 coat containing KDEL receptors bud o ER head 2 toward Golgi Low pH of Golgi allows KDEL receptor to aJach to EREresident proteins within Golgi 3 When secretory vesicles bud o Golgi COP1 coat KDEL receptors aJached to protein go w vesicles 4 When secretory vesicles meet fuse w ER membrane high pH of ER causes KDEL receptor protein to release missorted EREresident protein into ER lumen DATA DATA CBIO3400 CBIO3400 cis cis How cid 3 are cid 3 proteins cid 3 migrating cid 3 through cid 3 the cid 3 Golgi How do proteins migrate through the How cid 3 are cid 3 proteins cid 3 migrating cid 3 Golgi Compare contrast the 2 models through cid 3 the cid 3 Golgi Vesicular Transport Model Cisternal Progression Model trans trans Endocytosis Lysosomes DATA Lysosomes CBIO3400 morphologically heterogeneous Lysosomes cid 882 Morphological cid 3 hetrogenuous cid 882 Break cid 882 down cid 3 compartment cid 882 cid 882 Hydrolases cid 3 come cid 3 from cid 3 Golgi Intra cid 882 and cid 3 extracellular cid 3 cell cid 3 debris cid 3 breakdown compartment microorganisms cid 3 nutrient cid 3 production for intraE extracellular debris microorganisms nutrient producBon hydrolases come from Golgi but are only expressed in lower pH of lysosome recycling center of cell Autophagy DATA Lysosomes cid 3 substrates cid 3 are cid 3 delivered cid 3 via cid 3 three cid 3 routes CBIO3400 autophagy mitochondria digested if not funcBoning properly early late endosomes fuse w vesicles from Golgi Golgi vesicles bring hydrolyBc enzymes pH gets more more acidic from early endosome late endosome lysosome where hydrolyBc enzymes acBvate Lysosome cid 3 maturation How Do Lysosomal Hydrolases Get to the Lysosome Endosome CBIO3400 1 2 3 4 5 6 7 8 DATA M6P acts as sorBng signal is added in Golgi M6P receptors bind M6PEmodi ed proteins coat adaptors w clathrin coat GlcNAc phosphotransferase adds GlcNAcEPO4 to mannose in CGN GlcNAc removed in TGN leaving mannoseEPO4 Mannose cid 882 6 cid 882 phosphate M6P receptors bind lysosomal proteins in TGN where pH is 6 7 but must release added cid 3 in cid 3 the cid 3 Golgi cid 3 is cid 3 the cid 3 their cargo in late endosome where pH is sorting cid 3 signal 6 0 hence M6P receptor binding is pHEdependent Removal of PO4 from mannose makes transport unidirecBonal M6P thus becomes mannoseE6 which Cis cid 3 GlcNAc cid 882 phospho cid 882 cannot bind M6P receptors transferase 2a cid 3 2b cid 3 2g M6P receptors recycle back to TGN from early endosome using a retromer coat Two cid 882 step cid 3 process Trans cid 3 uncovering cid 3 enzyme cid 3 NAGPA cid 3 N cid 882 acetylglucosamine cid 882 1 cid 882 phosphodiester cid 3 alpha cid 882 N cid 882 acetylglucosaminidase cid 3 M6P cid 3 receptor cid 3 cycles cid 3 between Golgi cid 3 and cid 3 early cid 3 endosome M6P Biosynthesis Occurs via a 2QStep Pathway in the Golgi GlcNAcQ1Q phosphotransferase a heterohexameric enzymes encoded by GNPTAB GNPTG GNPTAB processed into subunits responsible for catalysis GNPTG encodes subunit funcBon unclear Mannose cid 3 6 cid 882 Phosphate cid 3 M6P cid 3 Biosynthesis cid 3 Occurs cid 3 Via cid 3 a Two cid 882 Step cid 3 Pathway cid 3 in cid 3 the cid 3 Golgi cid 882 P cid 882 cid 882 P cid 882 P cid 882 cid 882 P UDP cid