Presentation on theme: "Targeting and assembly of proteins destined for chloroplasts and mitochondria How are proteins targeted to chloroplasts and mitochondria from the cytoplasm?"— Presentation transcript:
1Targeting and assembly of proteins destined for chloroplasts and mitochondria How are proteins targeted to chloroplasts and mitochondria from the cytoplasm?How do they get through the membranes?
2Two types of cytosolic ribosomes: free and membrane-bound. They synthesize proteins with different destinations.Fig. 4.3, Buchanan et al.
31. Peptide domains for targeting to different organelles Targeting DomainOrganelleERSignal peptide (SP)ChloroplastTransit peptide (TP)MitochondrionPre-sequenceNucleusNuclear localization signal (NLS)PeroxisomePeroxisomal targeting signal(s) (PTS1 and PTS2)VacuoleVacuolar sorting signal (VSS)PTS1 is at the C-terminus; PTS2 is at the N-terminus.VSSs can be near or in the N-terminal signal peptide, or can be C-terminal peptides.
42. Chaperonins play roles in membrane transport on both sides of the membrane. Chaperones: Hsp70 typeFig. 4.2 Buchanan et al.
5Transport into organelles can be carried out in cell-free systems using in vitro-synthesized precursors.SS- rbcSPre-SS is about 24 kDa, and mature SS is about 18 kDa.Fig. 4.5, Buchanan et al.
6Maturation intermediate seen mainly with proteins destined for the inner (i.e., thylakoid membrane and lumen) compartments
7Features of chloroplast protein import (into the organelle) Post-translationalProteins synthesized as precursors with an Amino (N)-terminal extension.The N-terminal extension acts as the “zip code”, and often called “transit peptide”. It is removed during or soon after import.
84. Chaperonins bind to precursor before, during and after membrane translocation. Hsp70-type chaperonins maintain partially folded state in cytoplasm, whereas Hsp60 (cpn60) and Hsp70 promote folding inside organelle.5. ATP and GTP are also required for envelope membrane translocation.
96. Import receptors and translocation complexes (i. e 6. Import receptors and translocation complexes (i.e., Tocs and Tics) assemble at envelope membrane contact sites.Proteins of the outer membrane complex are called Tocs159, 75 and 34 kDa (159 and 34 kDa proteins bind GTP)Toc75 is the main pore (a beta-barrel protein)HSP70 IAP (or import intermediate associated protein) - functions between IM and OM)Inner membrane translocon complex proteins are called TicsTic20, Tic21, and Tic110 (kDa) proteins form channel7. After import, specific endoproteases in stroma remove transit peptide sequences.The 75 kDa protein is the pore, and is unusual as it is a Beta-barrel proteins (spans membrane 7 times but with beta strand structures in stead of alpha helices.
11Targeting to inner chloroplast compartments: thylakoid membrane- spanning and lumen proteins Proteins destined to the inner compartments (i.e., thylakoid-membrane spanning and lumen proteins) have longer Transit Peptides with 2 zip codes.They are removed in two steps:cleave cleavePrecursor Intermediate Mature
12the first cleavage unmasks a second sorting signal (zip code) the intermediate goes to the inner compartmentthe second cleavage generates mature protein
13Bipartite TP on lumen-targeted protein. ChaperonesTocsTicsBipartite TP on lumen-targeted protein.Fig. 4.6, Buchanan et al.
143 pathways for protein targeting into and across thylakoid membranes (to lumen) 3 pathways, but may share some components:secA-dependentpH gradient-dependent (or Tat pathway)SRP-dependent
15SecA-Dependent Pathway Involves a soluble, secA (bacterial gene) homologuerequires ATPpH gradient stimulatesExamples of proteins transported this way:PlastocyaninOE33 : 33 kDa protein of the oxygen evolving component of PSII (OEC)Also requires other sec genes besides secA.
16OEC (or OE) proteins of PSII mediate water splitting: Found in thylakoid lumen Yamamoto, Plant Cell Physiol. 2001
17pH Gradient-dependent (or Tat) pathway Requires the pH gradient across thylakoid membrane (generated by photosynthesis)Examples of proteins transported by this pathway:OE24 and OE17 subunits of the OECTransit peptides of these proteins have twin- arginine (Tat) motif that is essential for transport across thylakoidsalso occurs in bacteriaSRRxFLK is the bacterial signal (consensus) peptide.
18SRP-Dependent Pathway Involves a signal recognition particle (SRP)-like protein (cSRP54)SRP occurs in prokaryotes and eukaryotes, where its composed of an RNA (7SL) and several proteinsGreen plant chloroplast SRP does not have an RNA subunitrequires GTPpH gradient stimulatesExamples of proteins transported by this pathwayLHCPs: light-harvesting chlorophyll proteins (cab genes)Most chloroplast SRPs don’t’ seem to have the RNA component like bacteria.
19Role of SRP and its receptor in targeting to ER Fig. 4.15, Buchanan et al.
20P. Jarvis (2008) New Phytol 179:257-285 Carbonic Anhydrase 1 is imported via the ER-Golgi system; this pathway may be an evolutionary remnant that was maintained for dual targeting (ER-Golgi and Chloroplasts) of a few proteins. The PDI of Chlamy that binds the psbA mRNA 5’ UTR is dual targeted to ER and chloroplasts.P. Jarvis (2008) New Phytol 179:
21Differences in Mitochondrial vs Chloroplast targeting/import Many similarities between mitochondrial and chloroplast targeting/import mechanisms, but also important differences:Mitochondria have 1 less membrane and 1 less soluble compartmentthe proteins in the mito. membrane import machinery are not homologous to the Toc or Tic proteinsimport into the mito. matrix requires an electrochemical potential across the IM
22Proteins targeted to multiple organelles There are many targeted to both chloroplasts and mitochondriaExample: Most (~18 out of 20) of the organellar aminoacyl-tRNA synthetases in Arabidopsis are targeted to both organellesSome proteins found in both the chloroplast and the ER (or Golgi)Some ER Golgi Chloroplast protein targeting (Carbonic anhydrase 1 of A.t.)Prominent in algae with a Chloroplast ER
23Bioinformatic Predictions of Protein Subcellular Locations from Sequences Target PPredicts whether protein is Chloro., Mito., Secreted (Signal pep.) or CytosolicSignal PPredicts whether protein has signal peptideChloroPPredicts whether protein has a Transit peptide(Locating proteins in the cell using TargetP, SignalP, and related tools O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen. Nature Protocols 2, )PsortMitoprot., Predotar