Post trans mod-and_protein sorting

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  • Affect functional annotation, subcellular localizations, transmembrane topology
  • Post trans mod-and_protein sorting

    1. 1. Post-translationalPost-translationalmodification of proteinsmodification of proteins
    2. 2. End of TranslationEnd of Translation
    3. 3. Post-translational modificationsPost-translational modifications►Post-translationalPost-translationalmodifications (PTM) are keymodifications (PTM) are keymechanisms tomechanisms to increaseincreaseproteomic diversityproteomic diversity: same: sameprotein can have different PTMprotein can have different PTMleading to different 3D-leading to different 3D-structures.structures.►The post-translationalThe post-translationalmodifications are used tomodifications are used toregulate cellular activityregulate cellular activity
    4. 4. Post-translational modifications (PTM)Post-translational modifications (PTM)
    5. 5. Covalent Protein ModificationCovalent Protein Modification PTMs occur at distinct amino acid side chains and arePTMs occur at distinct amino acid side chains and areadded by specific enzymes.added by specific enzymes. Covalent modifications regulate the activity of enzymes andCovalent modifications regulate the activity of enzymes andmany other proteins.many other proteins. Most modifications are reversible, such as phosphorylation,Most modifications are reversible, such as phosphorylation,methylation, acetylation, and ubiquitination.methylation, acetylation, and ubiquitination. Some modifications are not reversible, such as adding aSome modifications are not reversible, such as adding alipid or sugar group.lipid or sugar group. Modification occursModification occurs - Co-translationally: require particular aa sequence- Co-translationally: require particular aa sequencecontexts for recognition;contexts for recognition; - Post-translationally: require accessibility of the target- Post-translationally: require accessibility of the targetresidues on the surface of the proteinresidues on the surface of the protein
    6. 6. GlycosylationGlycosylation►This is the addition of a carbohydrate or sugar moiety toThis is the addition of a carbohydrate or sugar moiety toproteins and this ranges from simple monosaccharideproteins and this ranges from simple monosaccharidemodifications of nuclear transcription factors to the complexmodifications of nuclear transcription factors to the complexbranched polysaccharide chains of cell surface receptors.branched polysaccharide chains of cell surface receptors.►WHY ? Glycosylations are often required for correctWHY ? Glycosylations are often required for correctpeptide folding and can increase protein stability andpeptide folding and can increase protein stability andsolubility and protect against degradation.solubility and protect against degradation.►Sugars are added to Threonine, tyrosine and SerineSugars are added to Threonine, tyrosine and Serinethrough O-linkage, and Asparagine and Arginine throughthrough O-linkage, and Asparagine and Arginine throughN-linkage.N-linkage.
    7. 7. GlycosylationGlycosylation
    8. 8. PhosphorylationPhosphorylation►Phosphorylation is the addition of aPhosphorylation is the addition of aphosphate (PO4) group to a serine, tyrosinephosphate (PO4) group to a serine, tyrosineor threonine residue in a peptide chainor threonine residue in a peptide chain►It plays an important role in regulating manyIt plays an important role in regulating manyimportant cellular processes such as cellimportant cellular processes such as cellcycle, growth, apoptosis (programmed cellcycle, growth, apoptosis (programmed celldeath) and signal transduction pathways.death) and signal transduction pathways.
    9. 9. PhosphorylationPhosphorylationThe addition or removal of a phosphate group can alter proteinconformation (and therefore function) by locally altering the charge andhydrophobicity where it is added.
    10. 10. N-AcetylationN-Acetylation►This process involves the transfer of an acetylThis process involves the transfer of an acetylgroup to nitrogen of Lys (K)group to nitrogen of Lys (K)►It has both reversible and irreversibleIt has both reversible and irreversiblemechanisms.mechanisms.► Acetylation helps in protein stability,Acetylation helps in protein stability,protection of the N-terminus and the regulationprotection of the N-terminus and the regulationof protein-DNA interactions in the case ofof protein-DNA interactions in the case ofhistones.histones.
    11. 11. MethylationMethylation►Protein methylation typically takes place onProtein methylation typically takes place onarginine or lysine amino acid residues in thearginine or lysine amino acid residues in theprotein sequence.protein sequence.►Methylation of histones, a type of DNA bindingMethylation of histones, a type of DNA bindingprotein, can regulate DNA transcription.protein, can regulate DNA transcription.
    12. 12. LipidationLipidation►Lipidation attaches a lipid group, such as aLipidation attaches a lipid group, such as afatty acid, covalently to a protein.fatty acid, covalently to a protein.►In general, lipidation helps in membraneIn general, lipidation helps in membranelocalization and targeting signalslocalization and targeting signals►Myristoylation plays a role in membraneplays a role in membranetargetingtargeting
    13. 13. Example: Meristoylation of N-Example: Meristoylation of N-terminal Glycineterminal Glycine
    14. 14. UbiquitinationUbiquitination► The addition of ubiquitin (an 8kDaThe addition of ubiquitin (an 8kDapolypeptide consisting of 76polypeptide consisting of 76amino acid residues) linked to anamino acid residues) linked to anamine group of lysine in targetamine group of lysine in targetprotein via its C-terminal glycine.protein via its C-terminal glycine.► Poly-ubiquitinated proteins arePoly-ubiquitinated proteins aretargeted for destruction whichtargeted for destruction whichleads to component recycling andleads to component recycling andthe release of ubiquitin.the release of ubiquitin.
    15. 15. ProteolysisProteolysis► Proteolysis is the breaking apart of the peptideProteolysis is the breaking apart of the peptidebond in a protein which can happen anywhere inbond in a protein which can happen anywhere ina protein.a protein.►It is an irreversible reaction.It is an irreversible reaction.►Proteolysis is important as it removesProteolysis is important as it removesunassembled protein subunits and misfoldedunassembled protein subunits and misfoldedproteins.proteins.
    16. 16. Intracellular CompartmentsIntracellular Compartmentsand Protein Sortingand Protein Sorting
    17. 17. ►Protein targeting, or protein trafficking, is theProtein targeting, or protein trafficking, is themoving of proteins from their site ofmoving of proteins from their site ofsynthesis to the place where they aresynthesis to the place where they areneeded.needed.►To reach their final destination, they mayTo reach their final destination, they maymove through cell different cellmove through cell different cellcompartmentscompartments
    18. 18. Intracellular Compartments andIntracellular Compartments andProtein SortingProtein Sorting►Functionally distinct membrane bound organelles►10 billion proteins of 10,000-20,00 diff kinds►Complex delivery system of these protein
    19. 19. Compartmentalization of CellsCompartmentalization of CellsMembranesMembranes► Partition cellPartition cell► Important cellular functionsImportant cellular functions► Impermeable to most hydrophilic moleculesImpermeable to most hydrophilic molecules► contain transport proteins to import and export specific moleculescontain transport proteins to import and export specific molecules
    20. 20. Compartmentalization of CellsCompartmentalization of CellsAll Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles
    21. 21. Compartmentalization of CellsCompartmentalization of Cells
    22. 22. Compartmentalization of CellsCompartmentalization of Cells
    23. 23. Compartmentalization of CellsCompartmentalization of CellsMajor Organelles►Nucleus►Cytosol►ER►Golgi Apparatus►Mitochondria and Chloroplast►Lysosomes►Endosomes►Peroxisomes
    24. 24. Final destination of protein afterFinal destination of protein aftertheir synthesis ?their synthesis ?►All proteins begin being synthesized onAll proteins begin being synthesized onribosomes in the cytosol, except for the fewribosomes in the cytosol, except for the fewthat are synthesized on the ribosomes ofthat are synthesized on the ribosomes ofmitochondria.mitochondria.►Their final destination depends on theirTheir final destination depends on theiramino acid sequence, which can containamino acid sequence, which can containsorting signalssorting signals that direct their delivery tothat direct their delivery tolocations outside the cytosol.locations outside the cytosol.
    25. 25. Protein targetingProtein targeting
    26. 26. Protein targetingProtein targeting►Most proteins do not have a sorting signalMost proteins do not have a sorting signaland consequently remain in the cytosol asand consequently remain in the cytosol aspermanent residents.permanent residents.►Many proteins, have specific sorting signalsMany proteins, have specific sorting signalsthat direct their transport from the cytosol intothat direct their transport from the cytosol intothe nucleus, the ER, mitochondria orthe nucleus, the ER, mitochondria orperoxisomes;peroxisomes;►Sorting signals can also direct the transport ofSorting signals can also direct the transport ofproteins from the ER to other destinations inproteins from the ER to other destinations inthe cell.the cell.
    27. 27. Proteins Can Move BetweenProteins Can Move BetweenCompartments in Different WaysCompartments in Different Ways3 Types of TransportMechanisms1.Gated Transport2.Transmembranetransport:3.Vesicular transport :
    28. 28. 1. Gated Transport1. Gated Transport►InIn gated transportgated transport, the protein, the proteinmoves between the cytosol andmoves between the cytosol andnucleus.nucleus.►The transport occurs in bothThe transport occurs in bothdirectionsdirections
    29. 29. 2. Transmembrane Transport:2. Transmembrane Transport:►The transported protein molecule usuallyThe transported protein molecule usuallymust unfold to move as a snake through themust unfold to move as a snake through thetranslocator tunnel on the membrane.translocator tunnel on the membrane.► Examples: The transport of proteins fromExamples: The transport of proteins fromthe cytosol into the ER lumen or from thethe cytosol into the ER lumen or from thecytosol into mitochondria.cytosol into mitochondria.
    30. 30. 3. Vesicular transport :3. Vesicular transport :►For example the transfer of soluble proteinsFor example the transfer of soluble proteinsfrom the ER to the Golgi apparatus,from the ER to the Golgi apparatus,Transport from Golgi to lysosomeTransport from Golgi to lysosome
    31. 31. Sorting signalsSorting signals2 Types of Sorting Signals in Proteins1. Signal Sequence (signal recognition peptide, SPR)ocontinuous sequence of 15-60 aaoFor some protein it will be removed from the protein sequence after itreach it destinationoFor some protein it is not remove and is part of finished protein2. Signal PatchComposed by non-continous amino acid sequences but their 3Dstructure forms a signal patch
    32. 32. Function of Sorting signalsFunction of Sorting signals
    33. 33. Signal patches direct proteins to:Signal patches direct proteins to:1. nucleus1. nucleus2. lysosomes2. lysosomesSignal Sequences direct proteins toSignal Sequences direct proteins to::1. ER proteins possess N-terminal signal of 5-10 hydrophobic aa1. ER proteins possess N-terminal signal of 5-10 hydrophobic aa2. Mitochondria proteins have alternating + charged aa with2. Mitochondria proteins have alternating + charged aa withhydrophobic aahydrophobic aa3. Proxisomal proteins have 3 aa at C-terminus3. Proxisomal proteins have 3 aa at C-terminusSignal Sequences/PatchesSignal Sequences/PatchesDirect Proteins to Final DestinationDirect Proteins to Final Destination
    34. 34. 34In secreted protein signal peptideIn secreted protein signal peptideis cleaved after secretionis cleaved after secretionCleaved off by type I signal peptidase (SPase I)Cleaved off by type I signal peptidase (SPase I)
    35. 35. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and CytosolNuclear Envelope►Two concentric membranes-Outer membrane contiguous w/ER-Inner membrane contains proteins that actabinding sites for chromatin and nuclear lamina►Perforated by nuclear pores for selectiveimport and export
    36. 36. ► reversiblereversible
    37. 37. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and CytosolNuclear Pore Complex►mass of 125 million; ~50 differentproteins arranged in octagon►Typical mammalian cell 3,000-4,000►Contains >1 aqueous channels thruwhich sm molec can readily pass<5,000; molec > 60,000 cannot pass►Functions ~diaphram►Receptor proteins actively transportmolec thru nuclear pore
    38. 38. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol
    39. 39. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and CytosolNuclear Localization SignalNuclear Localization Signal► Generally comprised of two short sequences rich in + chged aa lys & argGenerally comprised of two short sequences rich in + chged aa lys & arg► Can be located anywhereCan be located anywhere► Thought to form loops or patches on protein surfaceThought to form loops or patches on protein surface► The signal is not cleaved after the transportThe signal is not cleaved after the transport► Transport thru large aqueous poresTransport thru large aqueous pores► Transports proteins in folded stateTransports proteins in folded state► Energy requiring processEnergy requiring process
    40. 40. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and CytosolImport Receptors release cargo in nucleus and return to cytosolImport Receptors release cargo in nucleus and return to cytosolExport Receptors release cargo in cytoplasm and return to nucleusExport Receptors release cargo in cytoplasm and return to nucleus
    41. 41. Protein Transport into theProtein Transport into theMitochondria and ChloroplastMitochondria and ChloroplastSubcompartments of the Mitochondria and Chloroplast
    42. 42. Protein Transport into theProtein Transport into theMitochondria and ChloroplastMitochondria and ChloroplastTranslocation into Mitochondrial Matrix Governed by:1. Signal Sequence (amphipathic alpha helix cleaved after import)2. Protein Translocators
    43. 43. Protein Transport into theProtein Transport into theMitochondriaMitochondriaPlayers in Protein Translocation of Proteins in MitochondriaPlayers in Protein Translocation of Proteins in Mitochondria► TOM- functions across outer membraneTOM- functions across outer membrane► TIM- functions across inner membraneTIM- functions across inner membrane► OXA- mediates insertion of IM proteins syn w/in mito and helps toOXA- mediates insertion of IM proteins syn w/in mito and helps toinsert proteins initially transported into matrixinsert proteins initially transported into matrixComplexes contain components that act as receptors andComplexes contain components that act as receptors andothers that form translocation channelsothers that form translocation channels
    44. 44. Protein Transport into theProtein Transport into theMitochondriaMitochondriaImport of Mitochondrial Proteins►Post-translational►Unfolded polypeptide chain1. precursor proteins bind to receptor proteins of TOM2. interacting proteins removed and unfolded polypetide is fed intotranslocation channel►Occurs contact sites joining IM and OMTOM transports mito targeting signal across OM and once it reaches IMtargeting signal binds to TIM, opening channel complex thru which protein entersmatrix or inserts into IM
    45. 45. Protein Transport into theProtein Transport into theMitochondria and ChloroplastMitochondria and ChloroplastImport of Mitochondrial Proteins►Requires energy in form of ATP and H+ gradient and assitance of hsp70-release of unfolded proteins from hsp70 requires ATP hydrolysis-once thru TOM and bound to TIM, translocation thru TIM requireselectrochemical gradient
    46. 46. Protein Transport into theProtein Transport into theMitochondria and ChloroplastMitochondria and ChloroplastProtein Transport into IM or IM Space Requires 2 Signal Sequences1. Second signal =hydrophobic sequence; immediately after 1stsignal sequence2. Cleavage of N-terminal sequence unmasks 2ndsignal used to translocate proteinfrom matrix into or across IM using
    47. 47. ER and Protein TraffickingER and Protein TraffickingEndoplasmic ReticulumEndoplasmic Reticulum► Occupies >= 50% of cell volumeOccupies >= 50% of cell volume► Continuous with nuclear membraneContinuous with nuclear membrane► Central to biosyn macromolecules used to construct other organellesCentral to biosyn macromolecules used to construct other organelles► Trafficking of proteins to ER lumen, Gogli, lysosome or those to be secretedTrafficking of proteins to ER lumen, Gogli, lysosome or those to be secretedfrom cellfrom cell
    48. 48. ER and Protein TraffickingER and Protein TraffickingER Central to Protein Synthesis and Trafficking Removes 2 Types of Proteins from Cytosol:1. transmembrane proteins partly translocated across ER embedded in it2. water soluble proteins translocated into lumen
    49. 49. ER and Protein TraffickingER and Protein TraffickingImport of Proteins into ER►Occurs co-translationally►Signal recognition sequence recognized by SRP►SRP recognized by SRP receptor►Protein Translocator
    50. 50. ER and Protein TraffickingER and Protein Trafficking► Hydrophobic signal sequence of diff sequence and shapeHydrophobic signal sequence of diff sequence and shape► SRP lg hydrophobic pocket lined by Met having unbranched flexibleSRP lg hydrophobic pocket lined by Met having unbranched flexibleside chainsside chains► Binding of SRP causes pause in protein synthesis allowing time forBinding of SRP causes pause in protein synthesis allowing time forSRP-ribosome complex to bind to SRP receptorSRP-ribosome complex to bind to SRP receptor
    51. 51. ER and Protein TraffickingER and Protein TraffickingSome proteins are imported in to ER by a posttranslational mechanism►Proteins released into cytoplasm►Binding of chaperone proteins prevents them from folding
    52. 52. ER and Protein TraffickingER and Protein TraffickingSignal Sequence is Removed from Soluble ProteinsSignal Sequence is Removed from Soluble Proteins► Two signaling functions:Two signaling functions:1) directs protein to ER membrane1) directs protein to ER membrane2) serves as “start transfer signal” to open pore2) serves as “start transfer signal” to open pore► Signal peptidase removes terminal ER signal sequence uponSignal peptidase removes terminal ER signal sequence uponrelease of protein into the lumenrelease of protein into the lumen
    53. 53. ER and Protein TraffickingER and Protein TraffickingSingle Pass Transmembrane ProteinsSingle Pass Transmembrane Proteins1.1. N-terminal signal sequence initiates trans-N-terminal signal sequence initiates trans-location and additional hydrophobic “stoplocation and additional hydrophobic “stopsequence anchors protein in membranesequence anchors protein in membrane2.2. Signal sequence is internal and remains inSignal sequence is internal and remains inlipid bilayer after release from translocatorlipid bilayer after release from translocator3.3. Internal signal sequence in oppositeInternal signal sequence in oppositeorientationorientation4.4. Orientation of start-transfer sequenceOrientation of start-transfer sequencegoverned by distribution of nearby chg aagoverned by distribution of nearby chg aa
    54. 54. ER and Protein TraffickingER and Protein TraffickingMultipass Transmembrane ProteinsMultipass Transmembrane Proteins► Combinations of start- and stop-transfer signals determine topologyCombinations of start- and stop-transfer signals determine topology► Whether hydrophobic signal sequence is a start- or stop-transferWhether hydrophobic signal sequence is a start- or stop-transfersequence depends upon its location in polypeptide chainsequence depends upon its location in polypeptide chain► All copies of same polypeptide have same orientationAll copies of same polypeptide have same orientation
    55. 55. ER and Protein TraffickingER and Protein TraffickingFolding of ER Resident ProteinsFolding of ER Resident Proteins► ER resident proteins contain an ERER resident proteins contain an ERretention signal of 4 specific aa at C-retention signal of 4 specific aa at C-terminusterminus► PDI protein disulfide isomerase oxidizesPDI protein disulfide isomerase oxidizesfree SH grps on cysteines to from disulfidefree SH grps on cysteines to from disulfidebonds S-S allowing proteins to refoldbonds S-S allowing proteins to refold► BiP chaperone proteins, pulls proteinsBiP chaperone proteins, pulls proteinsposttranslationally into ER thru translocatorposttranslationally into ER thru translocatorand assists w/ protein foldingand assists w/ protein folding
    56. 56. ER and Protein TraffickingER and Protein TraffickingGlycolsylation of ER ProteinsGlycolsylation of ER Proteins► Most soluble and transmembrane proteins made in ER areMost soluble and transmembrane proteins made in ER areglycolsylated by addition of an oligosaccharide to Asnglycolsylated by addition of an oligosaccharide to Asn► Precursor oligosaccharide linked to dolichol lipid in ER mem, inPrecursor oligosaccharide linked to dolichol lipid in ER mem, inhigh energy statehigh energy state► Transfer by oligosaccharyl transferase occurs almost as soon asTransfer by oligosaccharyl transferase occurs almost as soon aspolypeptide enters lumenpolypeptide enters lumen
    57. 57. ER and Protein TraffickingER and Protein TraffickingRetrotranslocationRetrotranslocation► Improperly folded ER proteins are exported and degraded in cytosolImproperly folded ER proteins are exported and degraded in cytosol► Misfolded proteins in ER activate an “Unfolded Protein Response” toMisfolded proteins in ER activate an “Unfolded Protein Response” toincrease transcription of ER chaperones and degradative enzymesincrease transcription of ER chaperones and degradative enzymes
    58. 58. ENDEND

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