Importance of Protein sorting Cell organization depend on sorting ...

<strong>Importance</strong> <strong>of</strong> <strong>Protein</strong> <strong>sorting</strong><strong>Cell</strong> <strong>organization</strong> <strong>depend</strong> on <strong>sorting</strong>proteins to their right destination.<strong>Cell</strong> functions <strong>depend</strong> on <strong>sorting</strong> proteinsto their right destination.Examples:A. Energy production by mitochondriab. Transcriptional regulation:import <strong>of</strong> proteins, export <strong>of</strong> RNAc. Biogenesis <strong>of</strong> ER and Golgi,and proper functioning <strong>of</strong> the secretory systemd. Signal transduction networksQ: What is the relationship <strong>of</strong>intracellular compartments with oneanother?What is their evolutionary origin?A clue from plastiddevelopment12-3. Development <strong>of</strong>proplastid to differentiatedplastid [, e.g. chloroplast]involves membraneinvagination.12-4.Hypotheticalmodel for origin<strong>of</strong> organelles12-5. Topological relationships <strong>of</strong> compartments.Note: lumen = exterior <strong>of</strong> cellHow do proteins move to their destination?Membrane can bud and fuse. Vesicular transport12-6. Roadmap <strong>of</strong> proteintraffic.All proteins are made in thecytosol.Their fate <strong>depend</strong>s on the<strong>sorting</strong> signals.3 types <strong>of</strong> protein transport.12-8. Sorting signals built into a proteinWhat determines thedestination?Complementary <strong>sorting</strong> receptors recognize thesesignals.1

17-1. Sorting <strong>of</strong> nuclear encoded proteinsAtCNX: 1 MRQRQLFSVF LLLLAFVSFQ KLCYCDDQTV LYESFDEPFDER Calnexin, type ICytosolsolubleperipheralCytosolicRibosomes<strong>Protein</strong>Synthesis- cytosolOrganellar:Nuc: solubleMitoch: sol +membChloroplast: sol +membraneER-bound Ribosomes->secreted protein or membrane proteinsSoluble:ER lumenGolgi lumenVac lumenExtracellularNuc Env lumenMembrane:ERGolgiNucPMVacMOCB 639, Lodish 2000, ch. 17-1, 17-2; Alberts-ch 12Synthesis and <strong>sorting</strong> <strong>of</strong> nuclear-encoded proteins to organellesMajor questions1. How do proteins recognize their target destination?2. What is the identity <strong>of</strong> the molecules that recognize targeting information?3. How do large molecules pass through membranes? What is the driving force?4. What controls protein <strong>sorting</strong>?5. What approaches are used to study these questions?What lines <strong>of</strong> evidence support the model?Mitochondria: model <strong>of</strong> transmembrane transporta. Review <strong>of</strong> mitochondria structure, functionMost proteins coded by nuclear genes, synth in cyt, and imported.b. Method to study importc. Cyt Chaperones deliver proteins to mitod. Mito receptors transfer protein to channele. Import <strong>depend</strong>s on pmf and mito chaperones to keep proteins unfoldedf. Expt evidence for the model.Import into chloroplast16-7, 16-9. Mitochondrion2

17.3. Study protein import into mitochondria in a cell-free systemBiochemical approach = in vitroA. Label protein withisotope: In vitro synthesismRNA +35S-Metb. import assayFollow isotope-labeledprotein over time.Check protein is inside byprotease resistance.C. Test requirement forcytosolic factors or energyd. Test requirement formitochondria proteinswith mutant lacking amitoch. protein.Other approaches to study mechanism <strong>of</strong> translocationsee panel 12-1Transfection ApproachFind the <strong>sorting</strong> signal for mitochondria.Fuse targeting signal with cytosolic proteins.Genetic approache.g. yeast mutants defective in one protein <strong>of</strong> the uptake machinerycannot uptake mitochondria-destined proteinsSignal peptide is anamphipathic alpha helixwith no sequencehomology to othermito. Signals.Surface receptor andtranslocation pore form acomplexRecognition, insertion,translocation and processingEnergy is needed at 3 different steps:ATP and H+ gradient3

Repeated Hsp binding and ATP hydrolysis pullin proteinFig. 17-9. Chloroplast developmentand structureLight energy is used to oxidize water. Electrons are transferred toreduce NADPH and proton gradient is used to form ATP.CO 2 + RuBP -- rubisco--> 2 PGAPGA --NADPH, ATP --> G3P2G3P --> glucoseCF1FoTaiz + Zeiger (1998) Fig. 7-22Targeting proteins to the chloroplast:a. matrix Rubisco has single matrix signal sequenceb. thylakoid protein has 2.17-8. <strong>Protein</strong>s move into the thylakoid by one <strong>of</strong> four pathways.A. Sec ATP, ∆pH [PC, OEC33]b. SRP, GTP, ∆pH [LHCP]c. ∆pH [OE22, RR-p]d. Spontaneous [CFo-II]Keegstra K, Cline K. 1999.<strong>Protein</strong> import and routing systems <strong>of</strong> chloroplasts. Plant <strong>Cell</strong>. 11(4):557-70.4

Summary and a problemProblem: Do mito andchloroplast-destined proteins havedistinct matrix targetingsequences? Design anexperiment to test yourhypothesis.Evidence for matrix targeting signal Fig. 17-2Yeast ADH3p: 1 mlrtstlftr rvqpslfsrn ilrlqstaai pktqkgvify3-5 non-consecutive Arg(R) or Lys (K),with Ser (S) and Thr (T),no Asp (D) or Glu (E)<strong>Protein</strong> Import into mitochondrial matrixEvidence:1. Import <strong>depend</strong>s on cytosolic factors2. ATP is needed to keep protein unfolded3. Mitochondrial receptors are needed4. Import <strong>depend</strong>s on pmf and matrix chaperonespmf: provides a driving force17-4. <strong>Protein</strong> Import intomitochondrial matrixEvidence:1. Need for cytosolic factors2. cyt ATP is needed to keepprotein unfolded3. Mitochondrial receptors areneeded and being identified4. Import <strong>depend</strong>s on pmf andmatrix chaperones5