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

2. 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

3. 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!

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

5. DWD Proteins Tested members of each subgroup for DDB1 binding co-immunoprecipitation Two-hybrid: identifies interacting proteins

6. 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

7. 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!

8. 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

9. COP1 is a non-cullin-associated E3 ligase Recent data indicates that COP1 may also associate with CUL4

10. 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

11. 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

12. 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

13. 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

14. 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.

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

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

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

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

19. 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

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

21. PROTEIN TARGETING 2 Pathways in E.coli http://www.membranetransport.org/ 1.Tat: for periplasmic redox proteins & thylakoid lumen!

22. 2 Pathways in E.coli 1.Tat: for periplasmic redox proteins & thylakoid lumen! Preprotein has signal seqS/TRRXFLK

23. 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

24. 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

25. 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

26. 2 Pathways in E.coli http://www.membranetransport.org/ 1.Tat: for periplasmic redox proteins & thylakoid lumen! 2.Sec pathway SecB binds preprotein as it emerges from rib

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

28. 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

29. 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

30. 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

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

32. PROTEIN TARGETING Protein synthesis always begins on free ribosomes in cytoplasm

33. 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

34. 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

35. 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

36. 2 Protein Targeting pathways 1) Post -translational 2) Co-translational: Endomembrane system proteins are imported co-translationally inserted in RER as they are made

37. 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

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

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

40. 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

41. 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

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

43. 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

44. 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

45. 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

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

47. 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

48. Subsequent events Complications: proteins embedded in membranes

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

50. 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

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

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