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PROTEIN LOCALIZATION and SECRETION

PROTEIN LOCALIZATION and SECRETION. Collinearity. Order of nucleotides in the DNA gene would correlate with the order of amino acids in the corresponding polypeptide Charles Yanofsky showed this property using trpA, a gene that encodes a subunit of tryptophan synthetase

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PROTEIN LOCALIZATION and SECRETION

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  1. PROTEIN LOCALIZATION and SECRETION

  2. Collinearity Order of nucleotides in the DNA gene would correlate with the order of amino acids in the corresponding polypeptide Charles Yanofsky showed this property using trpA, a gene that encodes a subunit of tryptophan synthetase Mutated the gene and mapped it on the mutant protein DNA - stores the genetic information RNA - intermediate messenger Protein - functional component - all cellular activities --> complexity and diversity

  3. General Structure of L-amino acid

  4. Amino Acids

  5. Amino Acids

  6. Met NH2- COOH Polypeptide vs protein Linear non-branched polymer with an amino group and a carboxy group and R group side chain Posttranslational Modification Primary --> Secondary --> Tertiary --> Quaternary

  7. Levels of Secondary Structure -strand

  8. Levels of Secondary Structure - Another view

  9. Posttranslational Modification Removal or modification N-terminus or C-terminus amino acid residues fMet is often removed Modification of individual amino acids Phosphorylation of tyrosine (by kinases) Methylation Attachment of carbohydrate side chain Glycoproteins Trimming of polypeptides Holin of lambda phage Removal of signal sequence Discuss in detail Complexing with metal ions Metalloproteases

  10. NH2- COOH Met NH2- COOH Functional Domains DNA-binding Dimerization Protein is composed of series of structural and functional modules (motifs)

  11. Protein Localization and Secretion Prokaryotic Locations: 1. Cytoplasm 2. Cytoplasmic membrane 3. Periplasm Prokaryotic Secretions: 4. Extracellular 5. Into host cell

  12. Bacterial Membrane Gram (-) Gram (+) Lipoteichoic acid PG CM Cytoplasm Cytoplasm How do proteins translocate?

  13. Protein Localization and Secretion CM IM PG OM Memb CM Extra-cellular Bacterial Membranes Host Cell Membrane

  14. George E Palade Nobel Prize 1968 Gunther Blobel Nobel Prize 1999 Nobel Prizes • 1974 G Palade Med • “intracellular aspects of the process of protein secretion” • 1999 G Blobel Med • “for discovering that proteins have intrinsic signals that govern their transport and localization”

  15. Nobel Prize in Chemistry for 2003 • for discoveries concerning channels in cell membranes" "for the discovery of water channels" Peter Agre USA "for structural & mechanistic studies of ion channels" Roderick MacKinnon USA

  16. Bacterial Membrane

  17. Co-localization and Post-localization Translocon signal-recognition particle (SRP)

  18. Proteins crossing the membrane Fig 8.9

  19. Pos-translational folding/Chaperoned

  20. Pre-protein Pre-pro-protein Localization Signals Synthesizes secreted proteins with amino-terminal signal sequences as precursor protein - Pre-protein - Pre-pro-protein Pre- and pre-pro- proteins are escorted, either with or without the assistance of a chaperon protein Translocation can occur co-translationally or post-translationally No signals - but with un-cleaved signal anchor sequence - mostly IM proteins “Pre” - Signal sequence Three types of signal peptides (i) Standard Signal Peptide (ii) Lipoprotein Signal Peptide (iii) Type IV Prepilin Signal Peptide

  21. NH2- COOH Signal Peptide -1 +1 Cleavagesite -6 -5 -4 -3 -2 -1 +1 fM f G G G G G G G G G P A A P G G G S S -3 -2 -1 +1 +2 fM f G G G G G G G G L A C D G G Signal Peptides Neutral or Negative N H C 2-15 aa 2-3 charged K or R 14-20 aa Hydrophobic core; Rich in A & L Devoid of P, K, R, D, E, H and X 3-6 aa Standard Signal Peptide Lipoprotein Signal Peptide Modified C at +1 Lipoprotein signal +2

  22. NH2- COOH -3 -2 -1 +1 fM f f f f Q R G F G G G G E G G G K M Type IV Prepilin Signal Peptidase > 10 aa 2-3 charged K or R 14-20 aa Hydrophobic core; Rich in A & L Devoid of P, K, R, D, H and X -1 +1 Neutral or Negative N H Cleavagesite Methylated aa at +1 E at +6, G at +14 Info.bio.cmu.edu/Courses/03441/TermPapers/97TermPapers/Translocation/paper/signal_peptides.html

  23. Sample Signal Sequences Positively charged

  24. C A/G X L A/G X A/G G/P Periplasm IM/CM Cytoplasm Signal Peptidase Catalysis of the cleavage of N-terminal leader sequence for secreted and periplasmic precursor proteins Three peptidases F/M G R/K Q Signal Peptidase II (SPase II) / LspA Modified C at +1 L at -3 Signal Peptidase I (SPase I) / LepB G/P at -6 Prepilin Peptidase Cytoplasmic cleavage Methylated aa at +1

  25. Mutational Studies Synthesis-Folding-Localization What are the export routes? Using known genes that are exported Isolate secretion deficient mutants Map the genes Genes were named “sec” for genes involved in secretion Sec Pathways Sec-independent pathway Sequence analysis leading to signal peptide discovery

  26. Sec Pathways the Sec pathway transports proteins in an unfolded manner

  27. Simplified Sec System

  28. Twin Arginine Translocation Pathway Secretion of presumably folded cofactor-containing proteins across the IM Sec-independent Transports folded proteins and cofactors Cleavage of the N-terminal signal peptide by type I signal peptidase Signal peptide containing the characteristic S/TRRXFLK sequence Signal peptidase (membrane bound) cleavage site Ala-X-Ala specificity site

  29. Twin Arginine Transport - Signal Sequence Consensus Tat motif This and next 4 Figs from: Lee, Tullman-Ercek, Georgiou. 2006. The bacterial twin arginine translocation pathway. Ann Rev Microbiol 60:373-395

  30. Twin Arginine Transport (Tat)-Pathway The Tat pathway serves to actively translocate folded proteins across a lipidmembrane bilayer

  31. Hydrophobicity Plot --predicting membrane-spanning segments (highly hydrophobic) --exposed on the surface of proteins (hydrophilic domains) --potentially antigenic. A typical hydrophobicity plot of a protein with a signal sequence hydrophobic hydrophilic A typical hydrophobicity plot of a membrane protein

  32. Hydrophobicity Plot Amino Acid Characteristics – the R group Determines Everything

  33. Periplasm IM Cytoplasm PhoA/LacZ Fusion Analysis PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains.

  34. Bacteriorhodopsin

  35. Bacteriorhodopsin

  36. Glycophorin

  37. Protein Secretion CM IM PG OM Mem CM Extra-cellular Bacterial Cell Membrane Host Cell Membrane

  38. Types of Secretory Systems Sec (secretory) independent system (ABC-transporter-like) Type I Sec-dependent system (Type IV pilin-like) Type II Sec-independent system. Activated upon contact with host. Used by many pathogenic microbes. (Flagella-like) Type III Sec (??), also requires information inherent in the secreted protein itself. (Conjugative pili like) Type IV Sec-dependent system. Autotransporter System. The necessary information for trans-membrane negotiations inherent in the secreted protein itself. (Omp-like) Type V Phage spike like delivery system (Phage spike-like) “Hot off the press” Type VI

  39. Type I Secretion Pathway / General SP Sec-independent Completely bypass periplasm A complex of three proteins An inner membrane (IM) protein - an ATPase (ABC protein for ATP-binding cassette) A periplasmic space protein - anchored in the IM and spans the space to OM An outer membrane (OM) protein -”TolC” The targeting domain is carboxy terminus with a characteristic sequence variable number of copies of a glycine-rich repeat sequence Example: Secretion of Escherichia coli heamolysin (HlyA).

  40. A Model for Haemolysin (HlyA) Secretion in E. coli http://www.igmors.u-psud.fr/holland/haemolysin.html

  41. Type II / Sec-dependent System • By far the most common way used to translocate across the bacterial IM • Uses SPase I anchored to the periplasmic side of the IM • Several distinct components: • SRP - Signal recognition particle that binds to nascent peptide • SecA - A soluble, cytoplasmic, 102 kD homodimer; ATPase • SecB - A 17 kD homotetramer (Chaperon) • SecE (14 kD), G (15 kD), and Y (48 kD) • - A membrane channel • SecD and SecF • - may serve to aid in refolding • FtsY - docks SRP (not shown)

  42. Type II / Sec-dependent System Pre-proteins are escorted, either with or without the assistance of SecB Subsequently, an energy dependant reaction by SecA assists in the partial translocation Presumably through the SecYEG channel Signal sequence of the pre-protein remains embedded in the membrane until cleavage at the appropriate recognition site (typically Ala-X-Ala) by the Type I signal peptidase. The protein is transported out of periplasm through an outermembrane component called secretin

  43. Type III Secretion System Cytoplasmic delivery of proteins to host cells Secretion occurs in a continuous process without the distinct presence of periplasmic intermediates Does not involve proteolytic processing of secreted proteins A complex of approximately 20 proteins Most of them are located in the inner membrane Requires a cytoplasmic, probably membrane-associated ATPase Very similar to flagella biogenesis Molecular syringe Example: Tir/Intimin of Enteropathogenic Escherichia coli Yop proteins of Yersinia enterocolitica

  44. The Outer Membrane Secretin Family Gram-negative bacterial outer membrane proteins that form multimeric pores through which macromolecules, usually proteins, can pass. These proteins form homomultimeric ring structures, 10-20 subunits per complex, with large central pores (inner diameters of 50-100 Å). They are large proteins (420-750 amino acyl residues) Consisting of two domains: an N-terminal periplasmic domain and a C-terminal "homology" domain that is embedded in the OM exclusively responsible for channel formation. Secretins function in Type II and III protein secretion Example: In Vibrio cholerae, the secretin of the type III secretion system, EpsD, which exports cholera toxin

  45. Type III Secretion System

  46. Type III Secretion System Chromobacterium violaceum http://www.funpecrp.com.br/GMR/year2004/vol1-3/images/SCv0011fig2.jpg

  47. Intimin EPEC: Enteropathogenic Escherichia coli - Leading cause of bacterial mediated diarrhea in children First "harpoons" and embeds its receptor, called translocated intimin receptor (Tir) in the epithelial membrane of the host cell Then attaches to Tir via intimin; Form channel to deliver toxins 

  48. Type IV Secretion System Transport molecules toxic to host cells, but also are used to transport DNA or protein-DNA complexes. One such process is bacterial conjugation whereby two mating bacteria exchange genetic material. By facilitating conjugative transfer, type IV secretion machineries play crucial roles in the spread of antibiotic resistance genes among bacteria. Type IV secretion systems have been involved in pathogenicity caused by bacteria such as of Helicobacter pylori responsible for gastric ulcers or Legionella pneumophila responsible for Legionnaire disease.

  49. Type IV Secretion System VirB/VirD system of Agrobacterium tumefaciens has served as a prototype for T4SSs

  50. Bacteria genetically engineer plants with the Ti plasmid. Interaction of Ti plasmid with the plant genome Bacteria genetically engineer plants to control their differentiation (tumorigenic- Crown gall disease) and production of opines that can only be catabolized by the infecting Agrobacterium tumefaciens strain. Ti= tumor inducing

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