Post trans mod-and_protein sorting

Post-translational Post-translational modification of modification of

proteinsproteins

End of TranslationEnd of Translation

Post-translational Post-translational modificationsmodifications

►Post-translational Post-translational modifications (PTM) are key modifications (PTM) are key mechanisms to mechanisms to increase increase proteomic diversityproteomic diversity: same : same protein can have different protein can have different PTM leading to different 3D-PTM leading to different 3D-structures. structures.

►The post-translational The post-translational modifications are used to modifications are used to regulate cellular activityregulate cellular activity

Post-translational modifications (PTM)Post-translational modifications (PTM)

Covalent Protein Covalent Protein ModificationModification

PTMs occur at distinct amino acid side chains and PTMs occur at distinct amino acid side chains and are added by specific enzymes.are added by specific enzymes.

Covalent modifications regulate the activity of Covalent modifications regulate the activity of enzymes and many other proteins.enzymes and many other proteins.

Most modifications are reversible, such as Most modifications are reversible, such as phosphorylation, methylation, acetylation, and phosphorylation, methylation, acetylation, and ubiquitination.ubiquitination.

Some modifications are not reversible, such as Some modifications are not reversible, such as adding a lipid or sugar group.adding a lipid or sugar group.

Modification occursModification occurs - Co-translationally: require particular aa - Co-translationally: require particular aa

sequence contexts for recognition;sequence contexts for recognition; - Post-translationally: require accessibility of - Post-translationally: require accessibility of

the target residues on the surface of the the target residues on the surface of the protein protein

GlycosylationGlycosylation

►This is the addition of a carbohydrate or sugar This is the addition of a carbohydrate or sugar moiety to proteins and this ranges from simple moiety to proteins and this ranges from simple monosaccharide modifications of nuclear monosaccharide modifications of nuclear transcription factors to the complex branched transcription factors to the complex branched polysaccharide chains of cell surface receptors.polysaccharide chains of cell surface receptors.

►WHY ? Glycosylations are often required for WHY ? Glycosylations are often required for correct peptide folding and can increase protein correct peptide folding and can increase protein stability and solubility and protect against stability and solubility and protect against degradation.degradation.

►Sugars are added to Threonine, tyrosine and Sugars are added to Threonine, tyrosine and Serine through O-linkage, and Asparagine and Serine through O-linkage, and Asparagine and Arginine through N-linkage.Arginine through N-linkage.

Glycosylation Glycosylation

PhosphorylationPhosphorylation

►Phosphorylation is the addition of a Phosphorylation is the addition of a phosphate (PO4) group to a serine, phosphate (PO4) group to a serine, tyrosine or threonine residue in a tyrosine or threonine residue in a peptide chainpeptide chain

► It plays an important role in regulating It plays an important role in regulating many important cellular processes such many important cellular processes such as cell cycle, growth, apoptosis as cell cycle, growth, apoptosis (programmed cell death) and signal (programmed cell death) and signal transduction pathways.transduction pathways.

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.

N-AcetylationN-Acetylation►This process involves the transfer of an This process involves the transfer of an acetyl group to nitrogen of Lys (K) acetyl group to nitrogen of Lys (K) ►It has both reversible and irreversible It has both reversible and irreversible mechanisms. mechanisms. ► Acetylation helps in protein stability, Acetylation helps in protein stability, protection of the N-terminus and the protection of the N-terminus and the regulation of protein-DNA interactions in regulation of protein-DNA interactions in the case of histones.the case of histones.

MethylationMethylation►Protein methylation typically takes place Protein methylation typically takes place on arginine or lysine amino acid residues on arginine or lysine amino acid residues in the protein sequence.in the protein sequence.►Methylation of histones, a type of DNA Methylation of histones, a type of DNA binding protein, can regulate DNA binding protein, can regulate DNA transcription.transcription.

Lipidation Lipidation

►Lipidation attaches a lipid group, such Lipidation attaches a lipid group, such as a fatty acid, covalently to a protein. as a fatty acid, covalently to a protein. ►In general, lipidation helps in membrane In general, lipidation helps in membrane localization and targeting signalslocalization and targeting signals►Myristoylation plays a role in membrane plays a role in membrane targetingtargeting

Example: Meristoylation of N-Example: Meristoylation of N-terminal Glycineterminal Glycine

UbiquitinationUbiquitination

►The addition of ubiquitin (an The addition of ubiquitin (an 8kDa polypeptide consisting of 8kDa polypeptide consisting of 76 amino acid residues) linked 76 amino acid residues) linked to an amine group of lysine in to an amine group of lysine in target protein via its C-terminal target protein via its C-terminal glycine. glycine.

►Poly-ubiquitinated proteins are Poly-ubiquitinated proteins are targeted for destruction which targeted for destruction which leads to component recycling leads to component recycling and the release of ubiquitin. and the release of ubiquitin.

ProteolysisProteolysis

► Proteolysis is the breaking apart of the Proteolysis is the breaking apart of the peptide bond in a protein which can peptide bond in a protein which can happen anywhere in a protein. happen anywhere in a protein. ►It is an irreversible reaction. It is an irreversible reaction. ►Proteolysis is important as it removes Proteolysis is important as it removes unassembled protein subunits and unassembled protein subunits and misfolded proteins.misfolded proteins.

Intracellular Intracellular Compartments and Compartments and

Protein SortingProtein Sorting

►Protein targeting, or protein Protein targeting, or protein trafficking, is the moving of proteins trafficking, is the moving of proteins from their site of synthesis to the from their site of synthesis to the place where they are needed.place where they are needed.

►To reach their final destination, they To reach their final destination, they may move through cell different cell may move through cell different cell compartmentscompartments

Intracellular Compartments and Intracellular 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

Compartmentalization of Compartmentalization of CellsCells

MembranesMembranes► Partition cell Partition cell ► Important cellular functionsImportant cellular functions► Impermeable to most hydrophilic moleculesImpermeable to most hydrophilic molecules► contain transport proteins to import and export specific contain transport proteins to import and export specific

moleculesmolecules

Compartmentalization of CellsCompartmentalization of Cells

All Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles



Compartmentalization of Compartmentalization of CellsCells

Major Organelles

►Nucleus

►Cytosol

►ER

►Golgi Apparatus

►Mitochondria and Chloroplast

►Lysosomes

►Endosomes

►Peroxisomes

Final destination of protein Final destination of protein after their synthesis ?after their synthesis ?

►All proteins begin being synthesized All proteins begin being synthesized on ribosomes in the cytosol, except for on ribosomes in the cytosol, except for the few that are synthesized on the the few that are synthesized on the ribosomes of mitochondria.ribosomes of mitochondria.

►Their final destination depends on Their final destination depends on their amino acid sequence, which can their amino acid sequence, which can contain contain sorting signals sorting signals that direct their that direct their delivery to locations outside the delivery to locations outside the cytosol. cytosol.

Protein targetingProtein targeting

Protein targetingProtein targeting

►Most proteins do not have a sorting Most proteins do not have a sorting signal and consequently remain in the signal and consequently remain in the cytosol as permanent residents. cytosol as permanent residents.

►Many proteins, have specific sorting Many proteins, have specific sorting signals that direct their transport from signals that direct their transport from the cytosol into the nucleus, the ER, the cytosol into the nucleus, the ER, mitochondria or peroxisomes; mitochondria or peroxisomes;

►Sorting signals can also direct the Sorting signals can also direct the transport of proteins from the ER to transport of proteins from the ER to other destinations in the cell.other destinations in the cell.

Proteins Can Move Between Proteins Can Move Between Compartments in Different WaysCompartments in Different Ways

3 Types of Transport Mechanisms

1.Gated Transport

2.Transmembrane transport:

3.Vesicular transport :

1. Gated Transport1. Gated Transport

►In In gated transportgated transport, the protein , the protein moves between the cytosol and moves between the cytosol and nucleus. nucleus.

►The transport occurs in both The transport occurs in both directionsdirections

2. Transmembrane 2. Transmembrane Transport: Transport:

►The transported protein molecule The transported protein molecule usually must unfold to move as a snake usually must unfold to move as a snake through the translocator tunnel on the through the translocator tunnel on the membrane.membrane.

► Examples: The transport of proteins Examples: The transport of proteins from the cytosol into the ER lumen or from the cytosol into the ER lumen or from the cytosol into mitochondria.from the cytosol into mitochondria.

3. Vesicular transport :3. Vesicular transport :►For example the transfer of soluble For example the transfer of soluble

proteins from the ER to the Golgi proteins from the ER to the Golgi apparatus, Transport from Golgi to apparatus, Transport from Golgi to lysosomelysosome

Sorting signals Sorting 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

Function of Sorting signals Function of Sorting signals

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 1. ER proteins possess N-terminal signal of 5-10 hydrophobic aahydrophobic aa

2. Mitochondria proteins have alternating + charged 2. Mitochondria proteins have alternating + charged aa with aa with hydrophobic aahydrophobic aa

3. Proxisomal proteins have 3 aa at C-terminus3. Proxisomal proteins have 3 aa at C-terminus

Signal Sequences/Patches Signal Sequences/Patches Direct Proteins to Final DestinationDirect Proteins to Final Destination

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In secreted protein signal In secreted protein signal peptide is cleaved after peptide is cleaved after

secretionsecretion

Cleaved off by type I signal peptidase (SPase Cleaved off by type I signal peptidase (SPase I)I)

Transport of Molecules Btwn Nucleus and Transport of Molecules Btwn Nucleus and CytosolCytosol

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

► reversiblereversible


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



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

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 cytosolExport Receptors release cargo in cytoplasm and return to nucleusExport Receptors release cargo in cytoplasm and return to nucleus

Protein Transport into the Protein Transport into the Mitochondria and ChloroplastMitochondria and Chloroplast

Subcompartments of the Mitochondria and Chloroplast


Translocation into Mitochondrial Matrix Governed by:

1. Signal Sequence (amphipathic alpha helix cleaved after import)

2. Protein Translocators

Protein Transport into the Protein Transport into the MitochondriaMitochondria

Players in Protein Translocation of Proteins in Players in Protein Translocation of Proteins in MitochondriaMitochondria

► 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 OXA- mediates insertion of IM proteins syn w/in mito and helps

to insert proteins initially transported into matrixto insert proteins initially transported into matrix

Complexes contain components that act as receptors and Complexes contain components that act as receptors and others that form translocation channelsothers that form translocation channels

Protein Transport into the Protein 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


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


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

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 Trafficking of proteins to ER lumen, Gogli, lysosome or those to be

secreted from cellsecreted from cell


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


Import of Proteins into ER

►Occurs co-translationally

►Signal recognition sequence recognized by SRP

►SRP recognized by SRP receptor

►Protein Translocator


► Hydrophobic signal sequence of diff sequence and shapeHydrophobic signal sequence of diff sequence and shape► SRP lg hydrophobic pocket lined by Met having unbranched SRP lg hydrophobic pocket lined by Met having unbranched

flexible side chainsflexible side chains► Binding of SRP causes pause in protein synthesis allowing Binding of SRP causes pause in protein synthesis allowing

time for SRP-ribosome complex to bind to SRP receptortime for SRP-ribosome complex to bind to SRP receptor


Some proteins are imported in to ER by a posttranslational mechanism

►Proteins released into cytoplasm

►Binding of chaperone proteins prevents them from folding

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 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 Signal peptidase removes terminal ER signal sequence

upon release of protein into the lumenupon release of protein into the lumen


Single Pass Transmembrane ProteinsSingle Pass Transmembrane Proteins

1.1. N-terminal signal sequence initiates N-terminal signal sequence initiates trans-location and additional trans-location and additional hydrophobic “stop sequence anchors hydrophobic “stop sequence anchors protein in membraneprotein in membrane

2.2. Signal sequence is internal and remains Signal sequence is internal and remains in lipid bilayer after release from in lipid bilayer after release from translocatortranslocator

3.3. Internal signal sequence in opposite Internal signal sequence in opposite orientationorientation

4.4. Orientation of start-transfer sequence Orientation of start-transfer sequence governed by distribution of nearby chg governed by distribution of nearby chg aaaa


Multipass Transmembrane ProteinsMultipass Transmembrane Proteins► Combinations of start- and stop-transfer signals determine Combinations of start- and stop-transfer signals determine

topologytopology► Whether hydrophobic signal sequence is a start- or stop-Whether hydrophobic signal sequence is a start- or stop-

transfer sequence depends upon its location in polypeptide transfer sequence depends upon its location in polypeptide chainchain

► All copies of same polypeptide have same orientationAll copies of same polypeptide have same orientation


Folding of ER Resident ProteinsFolding of ER Resident Proteins► ER resident proteins contain an ER ER 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 PDI protein disulfide isomerase oxidizes free SH grps on cysteines to oxidizes free SH grps on cysteines to from disulfide bonds S-S allowing from disulfide bonds S-S allowing proteins to refoldproteins to refold

► BiP chaperone proteins, pulls proteins BiP chaperone proteins, pulls proteins posttranslationally into ER thru posttranslationally into ER thru translocator and assists w/ protein translocator and assists w/ protein foldingfolding

ER and Protein TraffickingER and Protein TraffickingGlycolsylation of ER ProteinsGlycolsylation of ER Proteins► Most soluble and transmembrane proteins made in ER are Most 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 Precursor oligosaccharide linked to dolichol lipid in ER

mem, in high energy statemem, in high energy state► Transfer by oligosaccharyl transferase occurs almost as Transfer by oligosaccharyl transferase occurs almost as

soon as polypeptide enters lumensoon as polypeptide enters lumen


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” Misfolded proteins in ER activate an “Unfolded Protein Response”

to increase transcription of ER chaperones and degradative to increase transcription of ER chaperones and degradative enzymesenzymes

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Technology

Post trans mod-and_protein sorting