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Protein targeting

Protein targeting. Lengeler et al. Chapter 19 pp. 484-489 Gene expression and regulatory mechanisms. Protein targeting. Stages in the pathway Cast of components Initial interactions Translocation apparatus. Protein targeting.

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Protein targeting

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  1. Protein targeting Lengeler et al. Chapter 19 pp. 484-489 Gene expression and regulatory mechanisms

  2. Protein targeting • Stages in the pathway • Cast of components • Initial interactions • Translocation apparatus

  3. Protein targeting • = translocation of newly synthesized polypeptides into / across biological membrane • Four stages • Recognition • Translocation • Proteolytic maturation • Release from export apparatus

  4. recognition • Leader sequences: ~20 to ~30 amino acids at N-terminus • +++ amino acids • Hydrophobic core • Signal sequence recognition sites: cleavage ↓ -XXXXX-↓-Cys-XXX---------- • lsp gene = lipoprotein-specific peptidase -XXXXAla-X-Ala-↓-XXX--------- • lep gene = leader peptidase

  5. Suppressor alleles • Normal “wild type” protein = good copy • Routed across CM • Recognized by export machinery • Correct processing, export • Mutant protein = defective copy • Not routed across CM; blocked • Recognized by export machinery only if its interacting partner protein also changes • Restores processing, export

  6. suppressors • Identification of interacting proteins • sec genes….secretion • Genetic mapping: E. coli chromosome • Non-essential components secB • Essential components secY, secE, secG secA secD, secF lepA, lspA

  7. Sec proteins • SecB • Soluble in cytoplasm • 16 kDa = octamer • Chaperone • Anti-folding activity • Recognizes polypeptide emerging from rib’s • Prevents folding into final conformation • SecB-dependent: maltose BP • SecB-independent: ribose BP

  8. Sec proteins • SecY • integral CM protein • highly hydrophobic • channel for passage of secreted proteins across CM • 3D structures: I. Collinson: cryo EM • SecE, SecG • Co-purify with SecY  complex

  9. Sec proteins • SecA • 2 forms: soluble; membrane-inserted • Recognizes [nascent polypeptide + SecB] • Escorts complex to SecY • ATPase activity: 2 binding domains • Piston action: sewing machine; threading

  10. proteolytic maturation • Polypeptide threaded through SecY • Emerges at periplasmic face of CM • Cleavage by LspA or LepA • Release of signal sequence • Folding of polypeptide in periplasm

  11. release • SecD • SecF • Liberate polypeptide from translocation cmplx  periplasm • Route polypeptide through adhesion sites • Contact regions between CM and OM • Export of proteins and LPS  OM

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