Targeting and assembly of proteins destined for chloroplasts and mitochondriaSlide 2Slide 3Slide 4Slide 5Slide 6Features of chloroplast protein import (into the organelle)Slide 8Slide 9Slide 10Targeting to inner chloroplast compartments: thylakoid membrane- spanning and lumen proteinsSlide 12Slide 133 pathways for protein targeting into and across thylakoid membranes (to lumen)SecA-Dependent PathwayOEC (or OE) proteins of PSII mediate water splitting: Found in thylakoid lumenpH Gradient-dependent (or Tat) pathwaySRP-Dependent PathwaySlide 19Slide 20Differences in Mitochondrial vs Chloroplast targeting/importProteins targeted to multiple organellesBioinformatic Predictions of Protein Subcellular Locations from SequencesTargeting 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?Two types of cytosolic ribosomes: free and membrane-bound.They synthesize proteins with different destinations. Fig. 4.3, Buchanan et al.ER Signal peptide (SP)Chloroplast Transit peptide (TP)Mitochondrion Pre-sequence Nucleus Nuclear localization signal (NLS)Peroxisome Peroxisomal targeting signal(s) (PTS1 and PTS2)Vacuole Vacuolar sorting signal (VSS)OrganelleTargeting Domain1. Peptide domains for targeting to different organelles2. Chaperonins play roles in membrane transport on both sides of the membrane.Fig. 4.2 Buchanan et al.Transport into organelles can be carried out in cell-free systems using in vitro-synthesized precursors.Fig. 4.5, Buchanan et al.SS- rbcSMaturation intermediate seen mainly with proteins destined for the inner (i.e., thylakoid membrane and lumen) compartmentsFeatures of chloroplast protein import (into the organelle)1. Post-translational 2. Proteins synthesized as precursors with an Amino (N)-terminal extension.3. The N-terminal extension acts as the “zip code”, and often called “transit peptide”. It is removed during or soon after import.4. 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.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 Tocs•159, 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 Tics• Tic20, Tic21, and Tic110 (kDa) proteins form channel 7. After import, specific endoproteases in stroma remove transit peptide sequences.Fig. 4.6, Buchanan et al.ChaperonesTocsTicsTargeting to inner chloroplast compartments: thylakoid membrane- spanning and lumen proteinsProteins 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 cleave Precursor  Intermediate  Mature•the first cleavage unmasks a second sorting signal (zip code)•the intermediate goes to the inner compartment•the second cleavage generates mature proteinFig. 4.6, Buchanan et al.ChaperonesTocsTicsBipartite TP on lumen-targeted protein.3 pathways for protein targeting into and across thylakoid membranes (to lumen)3 pathways, but may share some components:1. secA-dependent2. pH gradient-dependent (or Tat pathway) 3. SRP-dependentSecA-Dependent Pathway•Involves a soluble, secA (bacterial gene) homologue•requires ATP •pH gradient stimulates•Examples of proteins transported this way: –Plastocyanin–OE33 : 33 kDa protein of the oxygen evolving component of PSII (OEC)OEC (or OE) proteins of PSII mediate water splitting: Found in thylakoid lumen Yamamoto, Plant Cell Physiol. 2001pH 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 OEC •Transit peptides of these proteins have twin-arginine (Tat) motif that is essential for transport across thylakoids–also occurs in bacteriaSRP-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 proteins•Green plant chloroplast SRP does not have an RNA subunit•requires GTP•pH gradient stimulates•Examples of proteins transported by this pathway– LHCPs: light-harvesting chlorophyll proteins (cab genes)Fig. 4.15, Buchanan et al.Role of SRP and its receptor in targeting to ERP. Jarvis (2008) New Phytol 179:257-285Differences in Mitochondrial vs Chloroplast targeting/import•Many similarities between mitochondrial and chloroplast targeting/import mechanisms, but also important differences:1. Mitochondria have 1 less membrane and 1 less soluble compartment 2. the proteins in the mito. membrane import machinery are not homologous to the Toc or Tic proteins3. import into the mito. matrix requires an electrochemical potential across the IMProteins targeted to multiple organelles•There are many targeted to both chloroplasts and mitochondria–Example: Most (~18 out of 20) of the organellar aminoacyl-tRNA synthetases in Arabidopsis are targeted to both organelles•Some 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 ERBioinformatic Predictions of Protein Subcellular Locations from Sequences•Target P–Predicts whether protein is Chloro., Mito., Secreted (Signal pep.) or Cytosolic•Signal P–Predicts whether protein has signal peptide•ChloroP–Predicts whether protein has a Transit peptide •(Locating proteins in the cell using TargetP, SignalP, and related tools. 2007. O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen. Nature Protocols 2, 953-971)•Psort•Mitoprot.,