Quiz 3 Material Cytoskeleton MFs Ac1n Polymeriza1on8Based Mo1lity MFs Ac1n Motor Protein8Based Mo1lity MTs Ac1n8Myosin Systems MTs MT Motors Mitosis Mitosis Cytokinesis Cilia IFs Mitochondria Structure Genome Origins Mitochondria Protein Import Mitochondria Protein Import 2 Membranes 5 Protein Translocases Two Membranes Five Protein Translocases Figure 12 23 Molecular Biology of the Cell Garland Science 2008 mainly involved in import of proteins involved in mt genome thought to be derived from original endosymbiont gene encoded in mt genome not in nucleus Protein Import Pathways Protein Import Pathways 4 Des na ons 4 Destinations for proteins Summary of pathways Protein Import Pathways Summary proteins move across or insert into membranes of mitochondrion outer membrane TOM complex Translocase of Outer Membrane proteins go into intermembrane space SAM complex Sor1ng Assembly Machinery proteins go into outer membrane inner membrane TIM23 complex Translocase of Inner Membrane proteins go into matrix pre8sequence translocase TIM22 complex Translocase of Inner Membrane carrier translocase for carrier proteins targe1ng into matrix or membranes depends on di erent signal pep1des Outline of Matrix Import Outline of Protein Entry into Matrix Figure 12 25 Molecular Biology of the Cell Garland Science 2008 Receptors 2 Receptors TOM20 TOM70 signal pep1des determine receptor used TOM20 22 binds MTS Matrix Targe1ng Signal recognizes end8terminal signal TOM70 binds internal targe ng domains chaperones in cytoplasm cHSP70 HSP40 delivers pre8protein to TOM20 cHSP70 HSP90 delivers target membrane proteins to TOM70 Internal Targe ng Signals 1 HSP70 HSP90 bind hydrophobic pre8cursors 2 Cargo pre8protein delivered to TOM70 3 Mul1ple TOM70 molecules bind single cargo 4 TOM70 aggregates 5 In presence of ATP cargo transfers to TOM40 Internal Targeting Signals Hsp70 hsp90 bind to hydrophobic precursors Have multiple internal targeting signals Cargo preprotein is delivered to Tom70 Multiple Tom70 molecules bind to single cargo Tom70 aggregates In presence of ATP cargo is transferred to Tom40 TOM40 MTS and is receptor Matrix Targe ng Signal MTS 20830 AAs long forms amphipathic 8helix 8charged residues on one side bound by TOM22 hydrophobic uncharged residues on other side hydrophobic face bound by TOM20 aqracted by membrane poten1al Substrate PreIProtein Transfer to TOM40 general route TOM70 TOM20 22 Transfer of Preprotein to Tom40 pre8protein goes through TOM20 22 rst if it comes as aggregated blob then have to go through TOM70 rst TOM5 TOM40 relay of rela1vely weak MTS binding events General route Tom70 Relay of relatively weak MTS binding events Tom20 22 Tom40 Tom5 Tom40 TOM40 Complex contains TOM40 TOM5 TOM6 TOM7 TOM6 TOM7 important for assembly of TOM40 structure of TOM40 Ibarrel protein e g porins forms pore in outer membrane folded proteins cannot pass through i e those proteins that have been translated need help from chaperones before they can go into TOM40 b c folded protein can t t Exi ng TOM40 Protein Paths after exiting Tom40 Bolender et al 2008 Crossing IM Protein Transport across Inner Membrane Two Protein Complexes are Required Crossing Inner Membrane Protein Transport Across Inner Membrane Presequence Translocase Tim23 complex Presequence translocase Associated Motor PAM requires 2 protein complexes pre8sequence translocase TIM23 PAM Pre8Sequence Translocase8Associated Motor the woman makes it happen Tim23 Complex TIM23 Complex slide 1 of 2 TIM17 21 23 50 complex TIM50 binds cargo in intermembrane space directs cargo to intermembrane domain of TOM22 seals TIM23 pore in absence of pre8protein prevents ion leakage need membrane poten1al to open TIM23 pore TIM21 TIM17 binds TOM22 in intermembrane space outcompetes pre8protein promotes release of pre8protein from TOM22 binds PAM18 links pore to motor for transloca1on Tim23 Presequence translocation channel Pore diameter is 13 A 1 3 nm only fits one helix N terminal hydrophilic domain in inter membrane space Needed for high affinity binding to positively charged MTS TIM23 Complex slide 2 of 2 Tim23 Translocase Binds MTS Electrophoretic force MTS Tim23 interaction MTS membrane potential Membrane Potential Needed to open Tim23 pore Import motor TIM23 pre8sequence transloca1on channel pore can only t 1 8helix N8terminal hydrophilic domain in intermembrane space needed for high a nity binding to 8charged MTS TIM23 translocase binds MTS electrophore1c force MTS TIM23 interac1on MTS 8charged TIM23 8 8charged MTS membrane poten1al membrane poten1al needed to open TIM23 pore help protein go through Transloca on Import Motor PAM Complex Translocation Motor PAM Complex Components Tim44 Pam18 Pam16 mtHsp70 Mge1p components TIM44 PAM18 PAM16 mtHSP70 Mge1p mtHsp70 Pams mtHSP70 PAMs TIM44 PAM16 PAM18 binds TIM23 pre8sequence translocase docking sire for mtHSP70 membrane protein binds TIM23 binds PAM18 forms heterodimer PAM17 aids assembly of mtHSP70 J8domain protein s1mulates mtHSP70 ATPase binds TIM17 mtHSP70 binds TIM44 binds PAM18 associates w import pep1de cargo Mge1p mtHSP70 PreIProtein Import Motor mtHSP70 ATP binds TIM44 1 mtHSP70 ATP binds transloca1ng pre8protein 2 Hydrolyzes ATP to ADP 3 mtHSP70 ADP preIprotein dissociates from TIM44 4 Mge1p causes exchange of ADP for ATP 5 Bound pep1de released by mtHSP70 6 New molecule of ATP binds mtHSP70 7 mtHSP70 ATP binds TIM44 cycle repeats Mge1p mitochondrial grpE binds mtHSP70 ADP causes exchange of ADP for ATP on mtHSP70 facilitates pre8protein release Trapping Pulling How PAM Promotes Protein Import via trapping pulling How PAM Promotes Import of Protein By both i Trapping and ii Pulling trapping more passive any 1me protein oscillates up then down far enough mtHSP70 binds protein trapping it pulling more ac1ve actual conf change release aqachment pulling repeat of protein by mtHSP70 unfolding of pre8protein domains mtHSP70 mutant unable to mtHsp70 Ssc1 bind TIM44 fails to import protein w secondary structure Unfolding of preprotein domains mtHsp70 mutant unable to bind Tim44 fails to import protein with secondary structure Energy Energy Requirements Energy Requirements 1 Membrane potential Exerts electrophoretic force on MTS Required for Tim23 channel to open 2 ATP hydrolysis by HSP 70 Cytoplasmic Hsp 70 brings protein to TOM slide 6 7 mitochondria matrix mHSP70 Contributes chemical energy the import motor membrane poten1al exerts electrophore1c force on MTS required for TIM23 channel to open ATP