3. Post-translational modificationsPost-translational modifications
►Post-translationalPost-translational
modifications (PTM) are keymodifications (PTM) are key
mechanisms tomechanisms to increaseincrease
proteomic diversityproteomic diversity: same: same
protein can have different PTMprotein can have different PTM
leading to different 3D-leading to different 3D-
structures.structures.
►The post-translationalThe post-translational
modifications are used tomodifications are used to
regulate cellular activityregulate cellular activity
5. Covalent Protein ModificationCovalent Protein Modification
PTMs occur at distinct amino acid side chains and arePTMs occur at distinct amino acid side chains and are
added by specific enzymes.added by specific enzymes.
Covalent modifications regulate the activity of enzymes andCovalent modifications regulate the activity of enzymes and
many 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 a
lipid or sugar group.lipid or sugar group.
Modification occursModification occurs
- Co-translationally: require particular aa sequence- Co-translationally: require particular aa sequence
contexts for recognition;contexts for recognition;
- Post-translationally: require accessibility of the target- Post-translationally: require accessibility of the target
residues on the surface of the proteinresidues on the surface of the protein
6. GlycosylationGlycosylation
►This is the addition of a carbohydrate or sugar moiety toThis is the addition of a carbohydrate or sugar moiety to
proteins and this ranges from simple monosaccharideproteins and this ranges from simple monosaccharide
modifications of nuclear transcription factors to the complexmodifications of nuclear transcription factors to the complex
branched 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 correct
peptide folding and can increase protein stability andpeptide folding and can increase protein stability and
solubility and protect against degradation.solubility and protect against degradation.
►Sugars are added to Threonine, tyrosine and SerineSugars are added to Threonine, tyrosine and Serine
through O-linkage, and Asparagine and Arginine throughthrough O-linkage, and Asparagine and Arginine through
N-linkage.N-linkage.
8. PhosphorylationPhosphorylation
►Phosphorylation is the addition of aPhosphorylation is the addition of a
phosphate (PO4) group to a serine, tyrosinephosphate (PO4) group to a serine, tyrosine
or 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 many
important cellular processes such as cellimportant cellular processes such as cell
cycle, growth, apoptosis (programmed cellcycle, growth, apoptosis (programmed cell
death) and signal transduction pathways.death) and signal transduction pathways.
9. PhosphorylationPhosphorylation
The addition or removal of a phosphate group can alter protein
conformation (and therefore function) by locally altering the charge and
hydrophobicity where it is added.
10. N-AcetylationN-Acetylation
►This process involves the transfer of an acetylThis process involves the transfer of an acetyl
group to nitrogen of Lys (K)group to nitrogen of Lys (K)
►It has both reversible and irreversibleIt has both reversible and irreversible
mechanisms.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 regulation
of protein-DNA interactions in the case ofof protein-DNA interactions in the case of
histones.histones.
11. MethylationMethylation
►Protein methylation typically takes place onProtein methylation typically takes place on
arginine or lysine amino acid residues in thearginine or lysine amino acid residues in the
protein sequence.protein sequence.
►Methylation of histones, a type of DNA bindingMethylation of histones, a type of DNA binding
protein, can regulate DNA transcription.protein, can regulate DNA transcription.
12. LipidationLipidation
►Lipidation attaches a lipid group, such as aLipidation attaches a lipid group, such as a
fatty acid, covalently to a protein.fatty acid, covalently to a protein.
►In general, lipidation helps in membraneIn general, lipidation helps in membrane
localization and targeting signalslocalization and targeting signals
►Myristoylation plays a role in membraneplays a role in membrane
targetingtargeting
14. UbiquitinationUbiquitination
► The addition of ubiquitin (an 8kDaThe addition of ubiquitin (an 8kDa
polypeptide consisting of 76polypeptide consisting of 76
amino acid residues) linked to anamino acid residues) linked to an
amine group of lysine in targetamine group of lysine in target
protein via its C-terminal glycine.protein via its C-terminal glycine.
► Poly-ubiquitinated proteins arePoly-ubiquitinated proteins are
targeted for destruction whichtargeted for destruction which
leads to component recycling andleads to component recycling and
the release of ubiquitin.the release of ubiquitin.
15. ProteolysisProteolysis
► Proteolysis is the breaking apart of the peptideProteolysis is the breaking apart of the peptide
bond in a protein which can happen anywhere inbond in a protein which can happen anywhere in
a protein.a protein.
►It is an irreversible reaction.It is an irreversible reaction.
►Proteolysis is important as it removesProteolysis is important as it removes
unassembled protein subunits and misfoldedunassembled protein subunits and misfolded
proteins.proteins.
17. ►Protein targeting, or protein trafficking, is theProtein targeting, or protein trafficking, is the
moving of proteins from their site ofmoving of proteins from their site of
synthesis to the place where they aresynthesis to the place where they are
needed.needed.
►To reach their final destination, they mayTo reach their final destination, they may
move through cell different cellmove through cell different cell
compartmentscompartments
18. Intracellular Compartments andIntracellular Compartments and
Protein SortingProtein Sorting
►Functionally distinct membrane bound organelles
►10 billion proteins of 10,000-20,00 diff kinds
►Complex delivery system of these protein
19. Compartmentalization of CellsCompartmentalization of Cells
MembranesMembranes
► 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
24. Final destination of protein afterFinal destination of protein after
their synthesis ?their synthesis ?
►All proteins begin being synthesized onAll proteins begin being synthesized on
ribosomes in the cytosol, except for the fewribosomes in the cytosol, except for the few
that are synthesized on the ribosomes ofthat are synthesized on the ribosomes of
mitochondria.mitochondria.
►Their final destination depends on theirTheir final destination depends on their
amino acid sequence, which can containamino acid sequence, which can contain
sorting signalssorting signals that direct their delivery tothat direct their delivery to
locations outside the cytosol.locations outside the cytosol.
26. Protein targetingProtein targeting
►Most proteins do not have a sorting signalMost proteins do not have a sorting signal
and consequently remain in the cytosol asand consequently remain in the cytosol as
permanent residents.permanent residents.
►Many proteins, have specific sorting signalsMany proteins, have specific sorting signals
that direct their transport from the cytosol intothat direct their transport from the cytosol into
the nucleus, the ER, mitochondria orthe nucleus, the ER, mitochondria or
peroxisomes;peroxisomes;
►Sorting signals can also direct the transport ofSorting signals can also direct the transport of
proteins from the ER to other destinations inproteins from the ER to other destinations in
the cell.the cell.
27. Proteins Can Move BetweenProteins Can Move Between
Compartments in Different WaysCompartments in Different Ways
3 Types of Transport
Mechanisms
1.Gated Transport
2.Transmembrane
transport:
3.Vesicular transport :
28. 1. Gated Transport1. Gated Transport
►InIn gated transportgated transport, the protein, the protein
moves between the cytosol andmoves between the cytosol and
nucleus.nucleus.
►The transport occurs in bothThe transport occurs in both
directionsdirections
29. 2. Transmembrane Transport:2. Transmembrane Transport:
►The transported protein molecule usuallyThe transported protein molecule usually
must unfold to move as a snake through themust unfold to move as a snake through the
translocator tunnel on the membrane.translocator tunnel on the membrane.
► Examples: The transport of proteins fromExamples: The transport of proteins from
the cytosol into the ER lumen or from thethe cytosol into the ER lumen or from the
cytosol into mitochondria.cytosol into mitochondria.
30. 3. Vesicular transport :3. Vesicular transport :
►For example the transfer of soluble proteinsFor example the transfer of soluble proteins
from the ER to the Golgi apparatus,from the ER to the Golgi apparatus,
Transport from Golgi to lysosomeTransport from Golgi to lysosome
31. Sorting signalsSorting signals
2 Types of Sorting Signals in Proteins
1. Signal Sequence (signal recognition peptide, SPR)
ocontinuous sequence of 15-60 aa
oFor some protein it will be removed from the protein sequence after it
reach it destination
oFor some protein it is not remove and is part of finished protein
2. Signal Patch
Composed by non-continous amino acid sequences but their 3D
structure forms a signal patch
33. Signal patches direct proteins to:Signal patches direct proteins to:
1. nucleus1. nucleus
2. lysosomes2. lysosomes
Signal 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 aa
2. Mitochondria proteins have alternating + charged aa with2. Mitochondria proteins have alternating + charged aa with
hydrophobic aahydrophobic aa
3. Proxisomal proteins have 3 aa at C-terminus3. Proxisomal proteins have 3 aa at C-terminus
Signal Sequences/PatchesSignal Sequences/Patches
Direct Proteins to Final DestinationDirect Proteins to Final Destination
34. 34
In secreted protein signal peptideIn secreted protein signal peptide
is cleaved after secretionis cleaved after secretion
Cleaved off by type I signal peptidase (SPase I)Cleaved off by type I signal peptidase (SPase I)
35. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol
Nuclear Envelope
►Two concentric membranes
-Outer membrane contiguous w/ER
-Inner membrane contains proteins that act
abinding sites for chromatin and nuclear lamina
►Perforated by nuclear pores for selective
import and export
37. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol
Nuclear Pore Complex
►mass of 125 million; ~50 different
proteins arranged in octagon
►Typical mammalian cell 3,000-4,000
►Contains >1 aqueous channels thru
which sm molec can readily pass
<5,000; molec > 60,000 cannot pass
►Functions ~diaphram
►Receptor proteins actively transport
molec thru nuclear pore
38. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol
39. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol
Nuclear 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. Transport of Molecules Btwn Nucleus and CytosolTransport of Molecules Btwn Nucleus and Cytosol
Import Receptors release cargo in nucleus and return to cytosolImport Receptors release cargo in nucleus and return to cytosol
Export Receptors release cargo in cytoplasm and return to nucleusExport Receptors release cargo in cytoplasm and return to nucleus
41. Protein Transport into theProtein Transport into the
Mitochondria and ChloroplastMitochondria and Chloroplast
Subcompartments of the Mitochondria and Chloroplast
42. Protein Transport into theProtein Transport into the
Mitochondria and ChloroplastMitochondria and Chloroplast
Translocation into Mitochondrial Matrix Governed by:
1. Signal Sequence (amphipathic alpha helix cleaved after import)
2. Protein Translocators
43. Protein Transport into theProtein Transport into the
MitochondriaMitochondria
Players 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 to
insert proteins initially transported into matrixinsert proteins initially transported into matrix
Complexes contain components that act as receptors andComplexes contain components that act as receptors and
others that form translocation channelsothers that form translocation channels
44. Protein Transport into theProtein Transport into the
MitochondriaMitochondria
Import of Mitochondrial Proteins
►Post-translational
►Unfolded polypeptide chain
1. precursor proteins bind to receptor proteins of TOM
2. interacting proteins removed and unfolded polypetide is fed into
translocation channel
►Occurs contact sites joining IM and OM
TOM transports mito targeting signal across OM and once it reaches IM
targeting signal binds to TIM, opening channel complex thru which protein enters
matrix or inserts into IM
45. Protein Transport into theProtein Transport into the
Mitochondria and ChloroplastMitochondria and Chloroplast
Import 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 requires
electrochemical gradient
46. Protein Transport into theProtein Transport into the
Mitochondria and ChloroplastMitochondria and Chloroplast
Protein Transport into IM or IM Space Requires 2 Signal Sequences
1. Second signal =hydrophobic sequence; immediately after 1st
signal sequence
2. Cleavage of N-terminal sequence unmasks 2nd
signal used to translocate protein
from matrix into or across IM using
47. ER and Protein TraffickingER and Protein Trafficking
Endoplasmic 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 secreted
from cellfrom cell
48. ER and Protein TraffickingER and Protein Trafficking
ER Central to Protein Synthesis and Trafficking Removes 2 Types of Proteins from Cytosol:
1. transmembrane proteins partly translocated across ER embedded in it
2. water soluble proteins translocated into lumen
49. ER and Protein TraffickingER and Protein Trafficking
Import of Proteins into ER
►Occurs co-translationally
►Signal recognition sequence recognized by SRP
►SRP recognized by SRP receptor
►Protein Translocator
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 flexible
side chainsside chains
► Binding of SRP causes pause in protein synthesis allowing time forBinding of SRP causes pause in protein synthesis allowing time for
SRP-ribosome complex to bind to SRP receptorSRP-ribosome complex to bind to SRP receptor
51. ER and Protein TraffickingER and Protein Trafficking
Some proteins are imported in to ER by a posttranslational mechanism
►Proteins released into cytoplasm
►Binding of chaperone proteins prevents them from folding
52. ER and Protein TraffickingER and Protein Trafficking
Signal 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 membrane
2) 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 upon
release of protein into the lumenrelease of protein into the lumen
53. ER and Protein TraffickingER and Protein Trafficking
Single Pass Transmembrane ProteinsSingle Pass Transmembrane Proteins
1.1. N-terminal signal sequence initiates trans-N-terminal signal sequence initiates trans-
location and additional hydrophobic “stoplocation and additional hydrophobic “stop
sequence anchors protein in membranesequence anchors protein in membrane
2.2. Signal sequence is internal and remains inSignal sequence is internal and remains in
lipid bilayer after release from translocatorlipid bilayer after release from translocator
3.3. Internal signal sequence in oppositeInternal signal sequence in opposite
orientationorientation
4.4. Orientation of start-transfer sequenceOrientation of start-transfer sequence
governed by distribution of nearby chg aagoverned by distribution of nearby chg aa
54. ER and Protein TraffickingER and Protein Trafficking
Multipass 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-transfer
sequence 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. ER and Protein TraffickingER and Protein Trafficking
Folding of ER Resident ProteinsFolding of ER Resident Proteins
► ER resident proteins contain an ERER resident proteins contain an ER
retention signal of 4 specific aa at C-retention signal of 4 specific aa at C-
terminusterminus
► PDI protein disulfide isomerase oxidizesPDI protein disulfide isomerase oxidizes
free SH grps on cysteines to from disulfidefree SH grps on cysteines to from disulfide
bonds S-S allowing proteins to refoldbonds S-S allowing proteins to refold
► BiP chaperone proteins, pulls proteinsBiP chaperone proteins, pulls proteins
posttranslationally into ER thru translocatorposttranslationally into ER thru translocator
and assists w/ protein foldingand assists w/ protein folding
56. ER and Protein TraffickingER and Protein Trafficking
Glycolsylation of ER ProteinsGlycolsylation of ER Proteins
► Most soluble and transmembrane proteins made in ER areMost soluble and transmembrane proteins made in ER are
glycolsylated 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, in
high energy statehigh energy state
► Transfer by oligosaccharyl transferase occurs almost as soon asTransfer by oligosaccharyl transferase occurs almost as soon as
polypeptide enters lumenpolypeptide enters lumen
57. ER and Protein TraffickingER and Protein Trafficking
RetrotranslocationRetrotranslocation
► 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” to
increase transcription of ER chaperones and degradative enzymesincrease transcription of ER chaperones and degradative enzymes
