Quiz 2 Material Endomembrane System ER Protein Import ER Protein Folding Intracellular Vesicle Transport Golgi Endocytosis Lysosomes Exocytosis ER Protein Folding Heat Shock Proteins Hsp70 Hsp70 cid 3 heat cid 882 shock cid 3 protein cid 3 machinery cid 3 acts cid 3 early cid 3 in cid 3 the cid 3 life cid 3 of cid 3 many cid 3 proteins cid 3 by cid 3 binding cid 3 to cid 3 a cid 3 short cid 3 stretch cid 3 of cid 3 hydrophobic amino cid 3 acids cid 3 before cid 3 the cid 3 polypeptide cid 3 leaves cid 3 the cid 3 ribosome CBIO3400 DATA give proteins more Bme to reach their naBve conformaBon Hsp70 acts early in life of proteins by binding to a short stretch of hydrophobic AAs before polypepBde leaves ribosome binds hydrophobic region of Hsp70 System protein when protein starts to fold improperly MAIN GOAL to prevent protein aggregaBon giving them more Bme to nd correct conformaBon ATP hydrolysis provides energy used for protein folding Figure cid 3 6 cid 882 86 cid 3 Molecular cid 3 Biology cid 3 of cid 3 the cid 3 Cell cid 3 Garland cid 3 Science cid 3 2008 With cid 3 Hsp70 cid 3 the cid 3 main cid 3 goal cid 3 is cid 3 to cid 3 prevent cid 3 protein cid 3 aggregation cid 3 giving cid 3 them cid 3 more cid 3 time cid 3 to cid 3 find cid 3 the cid 3 correctly cid 3 folded cid 3 conformation HSP70 cid 3 proteins cid 3 are cid 3 found cid 3 in cid 3 cell cid 3 organelles cid 3 the cid 3 cytoplasm cid 3 and cid 3 the cid 3 ER Forma2on of Disordered Protein Aggregates is Relevant to Many Human Diseases Alzheimer s disease HunBngton s disease cysBc brosis are all characterized by defecBve folding aggregaBon of proteins DATA CBIO3400 Formation of Disordered Protein Aggregates is Relevant to Many Human Diseases Alzheimers disease cid 3 Huntington s cid 3 Disease cid 3 and cid 3 cystic cid 3 fibrosis cid 3 are cid 3 all cid 3 characterized cid 3 by cid 3 defective cid 3 folding cid 3 and cid 3 aggregation cid 3 of cid 3 proteins Glycosyla2on adding a sugar to AAs via a covalent bond postEtranslaBonal modi caBon associated w protein folding 2 types OQlinked happens on S or T NQlinked happens on asparagine NQLinked Glycosyla2on in the ER postEtranslaBonal modi caBon importance proper folding in ER aids protein funcBon 14Esugar residue precursor Glc 3 Man 9 GlcNAc 2 conserved in euks 90 of all glycoproteins modi ed in ER Golgi 5E residue core remains transfer to Asn via either AsnE XESer or AsnEXEThr DATA CBIO3400 N cid 882 linked cid 3 glycosylation cid 3 in cid 3 the cid 3 ER cid 882 synthesis cid 3 of cid 3 carbohydrate cid 3 chain cid 3 using cid 3 dolichol cid 882 P cid 3 and cid 3 activated cid 3 sugars cid 882 transfer cid 3 of cid 3 carbohydrate cid 3 chain cid 3 to cid 3 asparagine cid 3 of cid 3 protein cid 3 in cid 3 ER cid 882 processing cid 3 of cid 3 sugar cid 3 chain cid 3 ER cid 3 and cid 3 Golgi UDP cid 3 cid 882 Uridine cid 882 DP 14 cid 3 sugar cid 3 residue cid 3 precursor cid 3 Glc 3 Man 9 GlcNAc 2 conserved cid 3 in cid 3 eukaryotes cid 3 modified cid 3 5 cid 882 residue cid 3 core cid 3 remains NQLinked Glycosyla2on in the ER Tunicamycin QUESTION creates proteins that are less stable less acBve that cannot correctly interact w their binding partners blocks synthesis of all NEglycans CBIO3400 Uridine diphosphate N cid 882 acetylglucosamine tunicamycin Tunicamycin blocks cid 3 synthesis cid 3 of cid 3 all cid 3 N cid 882 glycans NQLinked Glycosyla2on in the ER OST Reac2on DATA N cid 882 linked cid 3 glycosylation cid 3 in cid 3 the cid 3 ER CBIO3400 cid 882 synthesis cid 3 of cid 3 carbohydrate cid 3 chain cid 3 using cid 3 dolichol cid 882 P cid 3 and cid 3 activated cid 3 sugars cid 882 transfer cid 3 of cid 3 carbohydrate cid 3 chain cid 3 to cid 3 asparagine cid 3 of cid 3 protein cid 3 in cid 3 ER cid 882 processing cid 3 of cid 3 sugar cid 3 chain cid 3 ER cid 3 and cid 3 Golgi Oligosaccharyl Transferase OST Reaction OST cid 882 an cid 3 ER cid 3 enzyme cid 882 Recognizes two cid 3 substrates Asn cid 882 X cid 882 Ser Thr on cid 3 protein Dolichol cid 882 P cid 882 P cid 882 GlcNAc2 cid 882 Man9 cid 882 Glc3 N cid 882 glycosylation cid 3 is a cid 3 protein cid 3 processing reaction posttranslational cid 3 modification sugar chain transferred mulBple Bmes by OST reac2on happens on ribosome b c enzyme must be present OST ER enzyme recognizes 2 substrates AsnEXESer or AsnEXEThr on protein dolicholEPEPE GlcNAc 2E Man 9E Glc 3 pyrophosphate on dolichol is hydrolyzed pushes sugar chain transfer sugar chain bound to residue aids protein folding b c it takes up a lot of space thus prevenBng AA interacBon within protein NQLinked Glycosyla2on in the ER 3 disBnct glycosidases remove the 3 glucoses oneEbyEone arer sugar chain is transferred to asparagine of ER protein 1 mannose removed sugar exits via specialized ER exits heads to cisEGolgi Man 8 GlcNAc 2 DATA CBIO3400 N cid 882 linked cid 3 glycosylation cid 3 in cid 3 the cid 3 ER cid 882 synthesis cid 3 of cid 3 carbohydrate cid 3 chain cid 3 using cid 3 dolichol cid 882 P cid 3 and cid 3 activated cid 3 sugars cid 882 transfer cid 3 of cid 3 carbohydrate cid 3 chain cid 3 to cid 3 asparagine cid 3 of cid 3 protein cid 3 in cid 3 ER cid 882 processing cid 3 of cid 3 sugar cid 3 chain cid 3 ER cid 3 and cid 3 Golgi UDP cid 3 cid 882 Uridine cid 882 DP 14 cid 3 sugar cid 3 residue cid 3 precursor cid 3 Glc 3 Man 9 GlcNAc 2 conserved cid 3 in cid 3 eukaryotes cid 3 modified cid 3 5 cid 882 residue cid 3 core cid 3 remains NQLinked Glycosyla2on in the ER Binding Protein BiP 2 domains ATPase domain NEterminal pepBde binding domain CEterminal ATP hydrolyzed to ADP BiP binds to protein prevenBng improper protein associaBon funcBons EREresident protein Hsp70 family member prevents aggregaBon of incorrectly folded proteins disaggregaBon of proteins binds to hydrophobic patches on proteins patches normally found on interior of proteins DATA CBIO3400 Calnexin and cid 3 calreticulin CNX CRT cid 3 are cid 3 ER cid 3 chaperones cid 3 that cid 3 help cid 3 glycoproteins cid 3 fold calnexin Ca2 cid 3 sugar cid 882 binding cid 3 and cid 3 protein cid 882 binding cid 3 …