transfer 5 µl from your pcr tube to fresh tube, add 1 µl dye & run on 0.7% gel
DESCRIPTION
Transfer 5 µl from your PCR tube to fresh tube, add 1 µl dye & run on 0.7% gel. Protein degradation Some have motifs marking them for polyubiquitination : E1 enzymes activate ubiquitin E2 enzymes conjugate ubiquitin E3 ub ligases determine specificity, eg for N-terminus. - PowerPoint PPT PresentationTRANSCRIPT
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Transfer 5 µl from your PCR tube to fresh tube, add 1 µl dye & run on 0.7% gel
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Protein degradationSome have motifs marking them for polyubiquitination:• E1 enzymes activate ubiquitin• E2 enzymes conjugate ubiquitin• E3 ub ligases determine specificity, eg for N-terminus
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E3 ubiquitin ligases determine specificity>1300 E3 ligases in Arabidopsis4 main classes according to cullin scaffolding protein• RBX positions E2• DDB1 positions DCAF/DWD• DCAF/DWD picks substrate• NOT4 is an E3 ligase & a component of the CCR4–NOT
de-A complex• CCR4–NOT de-A Complex regulates pol II• Transcription, mRNAdeg & prot deg are linked!
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins
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DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins•Only get transcription ifone hybrid supplies Act D& other supplies DNABinding Domain
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Regulating E3 ligasesThe COP9 signalosome (CSN), a complex of 8 proteins,
regulates E3 ligases by removing Nedd8 from cullinCAND1 then blocks cullinUbc12 replaces Nedd8Regulates DNA-damage response, cell-cycle & gene expressionNot all E3 ligases associate withCullins!
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COP1 is a non-cullin-associated E3 ligase• Protein degradation is important for light regulation• COP1/SPA1 tags transcription factors for degradation• W/O COP1 they act in dark• In light COP1 is exported to cytoplasm so TF can act
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COP1 is a non-cullin-associated E3 ligase• Recent data indicates that COP1 may also associate
with CUL4
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Protein degradation rate varies 100xMost have motifs marking them for polyubiquitination:
taken to proteosome & destroyedOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association with apoA-I) is degraded by calpain
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Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x
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Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub
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Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub• Recent yeast study doesn’t support general role
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Protein degradation rate varies 100xOther signals for selective degradation include PEST &
KFERQ• PEST : found in many rapidly degraded proteins• e.g. ABCA1 (which exports cholesterol in association
with apoA-I) is degraded by calpain• Deletion increases t1/2 10x, adding PEST drops t1/2 10x• Sometimes targets poly-Ub• Recent yeast study doesn’t support general role
• KFERQ: cytosolic proteins with KFERQ are selectively taken up by lysosomes in chaperone-mediated autophagy under conditions of nutritional or oxidative stress.
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Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon
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Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon2.Clp
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Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon2.Clp3.FtsH in IM
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PROTEIN TARGETINGAll proteins are made with an “address” which determines their final cellular location
Addresses are motifs within proteins
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PROTEIN TARGETINGAll proteins are made with “addresses” which determine their locationAddresses are motifs within proteins
Remain in cytoplasm unless contain information sending it elsewhere
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PROTEIN TARGETING Targeting sequences are both necessary & sufficient to send reporter proteins to new compartments.
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PROTEIN TARGETING2 Pathways in E.coli http://www.membranetransport.org/1.Tat: for periplasmic redox proteins & thylakoid lumen!
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2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK
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2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK•Make preprotein, folds & binds cofactor in cytosol
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2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK•Make preprotein, folds & binds cofactor in cytosol•Binds Tat in IM & is sent to periplasm
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2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK•Make preprotein, folds & binds cofactor in cytosol•Binds Tat in IM & is sent to periplasm•Signal seq is removed inperiplasm
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2 Pathways in E.coli http://www.membranetransport.org/1.Tat: for periplasmic redox proteins & thylakoid lumen!2.Sec pathway•SecB binds preproteinas it emerges from rib
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Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding
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Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding•Guides it to SecA, which drives it through SecYEG into periplasm using ATP
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Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding•Guides it to SecA, which drives it through SecYEG into periplasm using ATP•In periplasm signal peptide is removed and protein folds
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Sec pathway part deux•SRP binds preprotein as it emerges from rib & stops translation•Guides rib to FtsY•FtsY & SecA guide it to SecYEG , where it resumes translation & inserts protein into membrane as it is made
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Periplasmic proteins with the correct signals (exposed after cleaving signal peptide) are exported by XcpQ system
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PROTEIN TARGETINGProtein synthesis always begins on free ribosomes in cytoplasm
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2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes in cytoplasm1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationally
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2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes In cytoplasm1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationallymade in cytoplasm, then imported when complete
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2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes In cytoplasm1) Post -translational: proteins of plastids, mitochondria, peroxisomes and nuclei 2) Endomembrane system proteins are imported co-translationally
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2 Protein Targeting pathways1) Post -translational2) Co-translational: Endomembrane system proteins are imported co-translationallyinserted in RER as they are made
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2 pathways for Protein Targeting1) Post -translational2) Co-translational: Endomembrane system proteins are imported co-translationallyinserted in RER as they are madetransported to final destination in vesicles
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SIGNAL HYPOTHESISProtein synthesis always begins on free ribosomes in cytoplasmin vivo always see mix of free and attached ribosomes
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SIGNAL HYPOTHESISProtein synthesis begins on free ribosomes in cytoplasmendomembrane proteins have "signal sequence"that directs them to RER
Signal sequence
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SIGNAL HYPOTHESISProtein synthesis begins on free ribosomes in cytoplasmendomembrane proteins have "signal sequence"that directs them to RER“attached” ribosomes are tethered to RER by the signal sequence
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SIGNAL HYPOTHESIS• Protein synthesis begins on free ribosomes in cytoplasm• Endomembrane proteins have "signal sequence"that directs them to RER• SRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP
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SIGNAL HYPOTHESISSRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP•1 RNA & 7 proteins
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SIGNAL HYPOTHESISSRP binds signal sequence when it pops out of ribosome
SRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER
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SIGNAL HYPOTHESISSRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is made
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SIGNAL HYPOTHESISSRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is madeBiP (a chaperone) helps the protein fold in the lumen
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SIGNAL HYPOTHESISRibosome binds Translocon & secretes protein through it as it is madesecretion must be cotranslational
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Subsequent eventsSimplest case:
1) signal is cleaved within lumen by signal peptidase2) BiP helps protein fold correctly3) protein is soluble inside lumen
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Subsequent eventsComplications: proteins embedded in membranes
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proteins embedded in membranesprotein has a stop-transfer sequence
too hydrophobic to enter aqueous lumen
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proteins embedded in membranesprotein has a stop-transfer sequence
too hydrophobic to enter lumentherefore gets stuck in membraneribosome releases translocon, finishes job in cytoplasm
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More ComplicationsSome proteins have multiple trans-membrane domains (e.g. G-protein-linked receptors)
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More ComplicationsExplanation: combinations of stop-transfer and internal signals-> results in weaving the protein into the membrane