Transfer 5 µl from your PCR tube to fresh tube, add 1 µl dye & run on 0.7% gel

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

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!

DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation

DWD Proteins•Tested members of each subgroup for DDB1 binding • co-immunoprecipitation•Two-hybrid: identifiesinteracting proteins

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

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!

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

COP1 is a non-cullin-associated E3 ligase• Recent data indicates that COP1 may also associate

with CUL4

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

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

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

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

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.

Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon

Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon2.Clp

Protein degradation in bacteriaAlso highly regulated, involves chaperone like proteins1.Lon2.Clp3.FtsH in IM

PROTEIN TARGETINGAll proteins are made with an “address” which determines their final cellular location

Addresses are motifs within proteins

PROTEIN TARGETINGAll proteins are made with “addresses” which determine their locationAddresses are motifs within proteins

Remain in cytoplasm unless contain information sending it elsewhere

PROTEIN TARGETING Targeting sequences are both necessary & sufficient to send reporter proteins to new compartments.

PROTEIN TARGETING2 Pathways in E.coli http://www.membranetransport.org/1.Tat: for periplasmic redox proteins & thylakoid lumen!

2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!•Preprotein has signal seq S/TRRXFLK

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

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

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

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

Sec pathway•SecB binds preprotein as it emerges from rib & prevents folding

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

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

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

Periplasmic proteins with the correct signals (exposed after cleaving signal peptide) are exported by XcpQ system

PROTEIN TARGETINGProtein synthesis always begins on free ribosomes in cytoplasm

2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes in cytoplasm1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationally

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

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

2 Protein Targeting pathways1) Post -translational2) Co-translational: Endomembrane system proteins are imported co-translationallyinserted in RER as they are made

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

SIGNAL HYPOTHESISProtein synthesis always begins on free ribosomes in cytoplasmin vivo always see mix of free and attached ribosomes

SIGNAL HYPOTHESISProtein synthesis begins on free ribosomes in cytoplasmendomembrane proteins have "signal sequence"that directs them to RER

Signal sequence

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

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

SIGNAL HYPOTHESISSRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP•1 RNA & 7 proteins

SIGNAL HYPOTHESISSRP binds signal sequence when it pops out of ribosome

SRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER

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

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

SIGNAL HYPOTHESISRibosome binds Translocon & secretes protein through it as it is madesecretion must be cotranslational

Subsequent eventsSimplest case:

1) signal is cleaved within lumen by signal peptidase2) BiP helps protein fold correctly3) protein is soluble inside lumen

Subsequent eventsComplications: proteins embedded in membranes

proteins embedded in membranesprotein has a stop-transfer sequence

too hydrophobic to enter aqueous lumen

proteins embedded in membranesprotein has a stop-transfer sequence

too hydrophobic to enter lumentherefore gets stuck in membraneribosome releases translocon, finishes job in cytoplasm

More ComplicationsSome proteins have multiple trans-membrane domains (e.g. G-protein-linked receptors)

More ComplicationsExplanation: combinations of stop-transfer and internal signals-> results in weaving the protein into the membrane