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

E3 ubiquitin ligases determine specificity >1300 E3 ligases in Arabidopsis 4 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, mRNA deg & 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: identifies interacting proteins

DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation Two-hybrid: identifies interacting proteins Only get transcription if one hybrid supplies Act D & other supplies DNA Binding Domain

Regulating E3 ligases The COP9 signalosome (CSN), a complex of 8 proteins, regulates E3 ligases by removing Nedd8 from cullin CAND1 then blocks cullin Ubc12 replaces Nedd8 Regulates DNA-damage response, cell-cycle & gene expression Not all E3 ligases associate with Cullins!

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 100x Most have motifs marking them for polyubiquitination : taken to proteosome & destroyed Other 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 100x Other 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 t 1/2 10x, adding PEST drops t 1/2 10x

Protein degradation rate varies 100x Other 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 t 1/2 10x, adding PEST drops t 1/2 10x Sometimes targets poly-Ub

Protein degradation rate varies 100x Other 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 t 1/2 10x, adding PEST drops t 1/2 10x Sometimes targets poly-Ub Recent yeast study doesn’t support general role

Protein degradation rate varies 100x Other 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 t 1/2 10x, adding PEST drops t 1/2 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 bacteria Also highly regulated, involves chaperone like proteins 1.Lon

Protein degradation in bacteria Also highly regulated, involves chaperone like proteins 1.Lon 2.Clp

Protein degradation in bacteria Also highly regulated, involves chaperone like proteins 1.Lon 2.Clp 3.FtsH in IM

PROTEIN TARGETING All proteins are made with an “address” which determines their final cellular location Addresses are motifs within proteins

PROTEIN TARGETING All proteins are made with “addresses” which determine their location Addresses 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 TARGETING 2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen!

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

2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/TRRXFLK Make preprotein, folds & binds cofactor in cytosol

2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/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 seqS/TRRXFLK Make preprotein, folds & binds cofactor in cytosol Binds Tat in IM & is sent to periplasm Signal seq is removed in periplasm

2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! 2.Sec pathway SecB binds preprotein as 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 TARGETING Protein synthesis always begins on free ribosomes in cytoplasm

2 Protein Targeting pathways Protein synthesis always begins on free ribosomes in cytoplasm 1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationally

2 Protein Targeting pathways Protein synthesis always begins on free ribosomes In cytoplasm 1) proteins of plastids, mitochondria, peroxisomes and nuclei are imported post-translationally made in cytoplasm, then imported when complete

2 Protein Targeting pathways Protein synthesis always begins on free ribosomes In cytoplasm 1) Post -translational: proteins of plastids, mitochondria, peroxisomes and nuclei 2) Endomembrane system proteins are imported co-translationally

2 Protein Targeting pathways 1) Post -translational 2) Co-translational: Endomembrane system proteins are imported co-translationally inserted in RER as they are made

2 pathways for Protein Targeting 1) Post -translational 2) Co-translational: Endomembrane system proteins are imported co-translationally inserted in RER as they are made transported to final destination in vesicles

SIGNAL HYPOTHESIS Protein synthesis always begins on free ribosomes in cytoplasm in vivo always see mix of free and attached ribosomes

SIGNAL HYPOTHESIS Protein synthesis begins on free ribosomes in cytoplasm endomembrane proteins have "signal sequence"that directs them to RER Signal sequence

SIGNAL HYPOTHESIS Protein synthesis begins on free ribosomes in cytoplasm endomembrane 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 HYPOTHESIS SRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP 1 RNA & 7 proteins

SIGNAL HYPOTHESIS SRP binds signal sequence when it pops out of ribosome SRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER

SIGNAL HYPOTHESIS SRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is made

SIGNAL HYPOTHESIS SRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is made BiP (a chaperone) helps the protein fold in the lumen

SIGNAL HYPOTHESIS Ribosome binds Translocon & secretes protein through it as it is made secretion must be cotranslational

Subsequent events Simplest case: 1) signal is cleaved within lumen by signal peptidase 2) BiP helps protein fold correctly 3) protein is soluble inside lumen

Subsequent events Complications: proteins embedded in membranes

proteins embedded in membranes protein has a stop-transfer sequence too hydrophobic to enter aqueous lumen

proteins embedded in membranes protein has a stop-transfer sequence too hydrophobic to enter lumen therefore gets stuck in membrane ribosome releases translocon, finishes job in cytoplasm

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

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