Quiz 2 Material Endomembrane System ER Protein Import ER Protein Folding Intracellular Vesicle Transport Golgi Endocytosis Lysosomes Exocytosis ER Protein Import majority of proteins encoded in nucleus synthesized in cytoplasm then imported into organelles Isola2on of Cell Organelles Density Gradient Centrifuga2on METHOD Isolation cid 3 of cid 3 cell cid 3 organelles cid 3 by cid 3 density cid 3 gradient cid 3 centrifugation CBIO3400 get an equilibrium where cell density matches that of surrounding medium steps 1 HomogenizaBon 2 Get rough smooth microsomes Put microsomes in a tube w increasing sucrose gradient CentrifugaBon Smooth microsomes have low density closer to 5 rough microsomes have high density closer to 30 5 E30 sucrose gradients 4 5 3 molecules sediment according to their size shape density 30 5 METHOD CBIO3400 SDS cid 882 PAGE sodium cid 3 dodecyl cid 3 sulfate cid 3 polyacrylamide cid 3 gel cid 3 electrophoresis Separates cid 3 proteins cid 3 according cid 3 to cid 3 their cid 3 electrophoretic cid 3 mobility cid 3 SDS cid 3 anionic cid 3 detergent cid 3 to cid 3 linearize cid 3 proteins cid 3 and cid 3 adds cid 3 negative cid 3 charge cid 3 Charge cid 3 corresponds cid 3 cid 3 to cid 3 size all cid 3 proteins cid 3 have cid 3 the cid 3 same cid 3 mass cid 882 to cid 882 charge cid 3 ratio SDSQPAGE 2D cid 882 gel 1 cid 3 charge cid 3 no cid 3 SDS generates denatured protein w uniform neg charge separates proteins according to their electrophoreBc mobility uses SDS an anionic detergent to linearize proteins add neg charge charge corresponds to size mix detergent w proteins large proteins aJract a lot of SDS detergent thus developing a large charge indicates large protein size S D S l cid 3 s u p cid 3 cid 3 e z i s cid 3 2 CellQFree Transla2on System slide 1 of 3 ribosomes tRNAs ATP GTP mRNA for ER protein EXPERIMENT 1 METHOD Cell cid 882 free cid 3 translation cid 3 system cid 3 ribosomes cid 3 tRNAs cid 3 ATP cid 3 GTP cid 3 cid 3 mRNA cid 3 for cid 3 ER cid 3 protein cid 3 microsome A add protease A no protein microsome B run SDSQpage B protein protein B transported into microsome so CBIO3400 protected from protease treatment Experiment cid 3 1 cid 3 cid 882 microsomes A add cid 3 protease A cid 3 no cid 3 protein cid 3 cid 3 cid 3 microsomes B run cid 3 SDS cid 882 PAGE B cid 3 protein Experiment cid 3 2 as cid 3 exp cid 3 1 cid 3 but cid 3 no cid 3 protease cid 3 treatment Experiment cid 3 3 microsomes added cid 3 together cid 3 with cid 3 mRNA cid 3 A microsomes added cid 3 later cid 3 than cid 3 mRNA cid 3 B A B METHOD CBIO3400 Cell cid 882 free cid 3 translation cid 3 system cid 3 ribosomes cid 3 tRNAs cid 3 ATP cid 3 GTP cid 3 cid 3 mRNA cid 3 for cid 3 ER cid 3 protein cid 3 Experiment cid 3 1 cid 3 cid 882 microsomes A add cid 3 protease A cid 3 no cid 3 protein cid 3 cid 3 cid 3 microsomes B run cid 3 SDS cid 882 PAGE B cid 3 protein Experiment cid 3 2 CellQFree Transla2on System slide 2 of 3 as cid 3 exp cid 3 1 cid 3 but cid 3 no cid 3 protease cid 3 treatment EXPERIMENT 2 Experiment cid 3 3 microsomes added cid 3 together cid 3 with cid 3 mRNA cid 3 A microsomes added cid 3 later cid 3 than cid 3 mRNA cid 3 B same as E 1 except no protease treatment w SDSQPAGE signal sequence is cleaved o by speci c protease protein as it enters membrane protein size di erence protein A incubated w o organelle microsome protein B incubated w organelle microsome SDSQPAGE removes signal sequence from protein B so protein B is smaller A B METHOD Experiment cid 3 2 microsomes added cid 3 later cid 3 than cid 3 mRNA cid 3 B CellQFree Transla2on System CBIO3400 slide 3 of 3 EXPERIMENT 3 A microsomes added together w mRNA cellEfree protein synthesis microsomes present this process is coEtranslaBonal b c protein can enter microsome METHOD Experiment cid 3 2 microsomes added cid 3 later cid 3 than cid 3 mRNA cid 3 B B microsomes added later than mRNA cellEfree protein synthesis no microsomes present this process is postEtranslaBonal b c protein cannot reEenter microsome arer translaBon is CBIO3400 complete when you add microsomes later protein can no longer enter microsomes microsomes added cid 3 together cid 3 with cid 3 mRNA cid 3 A microsomes added cid 3 together cid 3 with cid 3 mRNA cid 3 A Signal Sequence 16E30 residues long hydrophobic stretch anked by posiBvelyE charged AAs not a sequence but a paJern required su cient to get into ER method test su ciency by adding signal sequence to any cytoplasmic protein watching it go into ER removed when present on NEterminus called signal anchor sequence when present internally no cleavage Signal Recogni2on Par2cle SRP Met cluster has hydrophobic binding groove changes conformaBon upon binding RNA 6 proteins ribonucleoprotein parBcle binds signal sequence pausing translaBon DATA CBIO3400 SRP Receptor SR SRP cid 3 Receptor cid 3 SR Heterodimer cid 882 SR cid 69 is cid 3 transmembrane protein cid 3 300 cid 3 aa cid 882 SR cid 68 is cid 3 a cid 3 peripheral membrane cid 3 protein cid 3 640 cid 3 aa cid 882 both cid 3 are cid 3 GTPases cid 882 Cytoplasmic cid 3 domain cid 3 of cid 3 SR cid 69 binds cid 3 SRP cid 3 and cid 3 ribosome and cid 3 relieves cid 3 block cid 3 in cid 3 chain cid 3 elongation heterodimer SR TM protein binds SRP relieves block in chain elongaBon can bind SRP or ribosome SR peripheral membrane protein both bind GTP are GTPases once an SRP has docked its SR on the membrane translaBon can begin again Translocon Sec61 Complex high a nity for ribosome receptor Chemical cid 3 cross cid 882 linking cid 3 revealed cid 3 translocon cid 882 nascent cid 3 protein cid 3 interactions CBIO3400 binds ribosome DATA 60S recognizes binds signal pepBde at ER membrane forms gated pore channel through lipid bilayer has a plug that can be removed when protein comes isoleucine creates a Bght seal w ribosome so not just anything can get through pore chemical crossElinking revealed translocon nascent protein interacBons TRAM translocaBng chainE associated membrane protein TRAM cid 3 cid 882 translocating chain cid 882 associated cid 3 membrane cid 3 protein ER Signal Pep2dase removes cleaves via endoproteolysis the signal sequence from the growing protein DATA CBIO3400 The cid 3 ER cid 3 signal cid 3 peptidase cid 3 removes cid 3 cleaves cid 3 by cid 3 endoproteolysis cid 3 the cid 3 signal …