Bridget, Jephte, Kristi, Matt, Teresa Set up 20 µl mix for each primer/DNA combo on ice!

1. Bridget, Jephte, Kristi, Matt, Teresa • Set up 20 µl mix for each primer/DNA combo on ice! • 2 µl 10x F primer (1 pMol/µl = 1µM final []) • 2 µl 10x R primer • 1 µl DNA • We will prepare Phusion master mix for 130 µl total volume • 26 µl 5x Phusion HF buffer • 2.6 µl 10 mM dNTP (200 µM final []) • 67.6 µl water • 1.3 µl Phusion polymerase • Add 15 µl master mix to each rxn • JKMT run at 58˚ C annealing T • Bridget run on touchdown starting at 72˚ C annealing T

2. Testing internal primers • Set up 20 µl mix for each primer/DNA combo on ice! • 2 µl 10x internal F primer (1 pMol/µl = 1µM final []) • 2 µl 10x internal R primer • 0.5 µl DNA = best PCR reaction • We will prepare master mix for 90 µl total volume • 9 µl 10x Taq buffer • 1.8 µl 10 mM dNTP (200 µM final []) • 58.4 µl water • 0.56 µl Taq polymerase • Add 15.5 µl master mix to each rxn • Run at 50˚ C annealing T, 2 minutes/72˚ cycle

3. 2 Protein Targeting pathways Protein synthesis always begins on free ribosomes In cytoplasm 1) Post -translational: proteins ofplastids,mitochondria, peroxisomesandnuclei 2) Endomembrane system proteins are imported co-translationally

4. Sorting proteins made on RER lysosomal proteins are targeted by mannose 6-phosphate M 6-P receptors in trans-Golgi direct protein to lysosomes (via endosomes) M 6-P is added in Golgi by enzyme that recognizes lysosomal motif

5. Glycosylation within ER All endomembrane proteins are highly glycosylatedon lumenal domains. Glycosylation starts in the ER, continues in the Golgi

6. Glycosylation within ER • All endomembrane proteins are highly glycosylatedon lumenal domains. • Glycosylation starts in ER, continues in Golgi • makes proteins more hydrophilic • essential for proper function • tunicamycin poisons cells • Glycosylation mutants are even sicker

7. Glycosylation in RER 1)(CH2O)n are assembled stepwise on dolichol-PO4 2) Transfer (CH2O)n to target asn

8. Glycosylation in RER 1)(CH2O)n are assembled stepwise on dolichol-PO4 2) Transfer (CH2O)n to target asn 3) remove 2 glucose & bind chaperone If good,remove gluc 3 & send to Golgi

9. Glycosylation in RER remove 2 glucose & bind to chaperone If good,remove gluc 3 & send to Golgi If bad, GT adds glucose & try again Eventually, send bad proteins to cytosol & eat them

10. Glycosylation • next modify (CH2O) n in Golgi • Remove some sugars & add others

11. Glycosylation • next modify (CH2O) n in Golgi • Remove some sugars & add others • different rxns occur in different parts of Golgi • why we separate Golgi into distinct regions

13. Post-translational protein targeting Key features 1) imported after synthesis

14. Post-translational protein targeting Key features 1) imported after synthesis 2) targeting information is motifs in protein a) which organelle b) site in organelle 3) Receptors guide it to correct site 4) no vesicles!

15. Protein targeting in Post-translational pathway • SKL (ser/lys/leu) at C terminustargets most peroxisomal matrix proteins = PTS1 • In humans 3 are targeted by 9 aa at N terminus = PTS2 • Defective PTS2 receptor causes Rhizomelic chondrodysplasia punctata N SKL C N PTS2 C

16. Targeting peroxisomal proteins • Bind receptor in cytoplasm • Dock with peroxisomal receptors • Import • protein w/o • unfolding it! • Recycle • receptors

17. Peroxisomal Membrane Synthesis Most peroxisomes arise by fission can arise de novo! Mechanism is poorly understood/ may involve ER! Only need PEX 3 & PEX 16 to import pex membrane prot

18. Protein import into nuclei • nuclear proteins are targeted by internal motifs • necessary & sufficient to target cytoplasmic proteins to nucleus

19. Protein import into nuclei • nuclear proteins are targeted by internal motifs • as in golgi, are not specific • shapes cf sequences • Receptors bind objects of the right shape!

20. Protein import into nuclei • 3 types of NLS (nuclear localization sequence) • 1) basic residues in DNA-binding region + + + LZ

21. Protein import into nuclei • 3 types of NLS (nuclear localization sequence) • 1) basic residues in DNA-binding region • 2) SV-40 KKKRK + + + LZ KKKRK

22. Protein import into nuclei • 3 types of NLS (nuclear localization sequence) • 1) basic residues in DNA-binding region • 2) SV-40 KKKRK • 3) bi-partite: 2-4 basic aa,10-20 aa spacer, 2-4 basic aa + + + LZ KKKRK + + + +

23. Protein import into nuclei • 1) importin-a binds NLS importin-b binds complex • 2) escort to nuclear pores • Pores decide who can enter/exit nucleus

24. Protein import into nuclei 1) importin-a binds NLS, importin-b binds complex 2) escort to nuclear pores 3) transporter changes shape, lets complex enter 4) nuclear Ran-GTP dissociates complex 5) Ran-GTP returns b-importinto cytoplasm, becomes Ran-GDP. GTP -> GDP = nuclear importenergy source 6) Exportins return a-importin& other cytoplasmic prot

25. Protein import into cp and mito • Many common features • 1) Pulse-chase experiments show most cp & mt proteins are made in cytoplasm as larger precursor (preprotein) • both have N-terminal targeting peptide • transit peptidein cp • presequence in mito • necessary & sufficient to target

26. Protein import into cp &mito • Many common features • 1) N-terminal transit peptideor presequence • necessary & sufficient to target • usually removed upon arrival

27. Protein import into cp & mito • Many common features • 1) N-terminal transit peptideor presequence • 2) both need energy input • a) ATP for both • b) Mt also use Proton Motive Force (PMF) • H+ gradient made by electron transport • c) Cp also use GTP (but not PMF)

28. Protein import into cp & mito • 1) N-terminal transit peptideor presequence • 2) both need energy input • 3) proteins unfold to enter, then refold inside • a) need chaperonins on both sides of membrane • i) chaperonins in cytosol unfold • ii) chaperonins inside refold • a) helps draw through membrane

29. Protein import into mitochondria • Precursor has N-terminal targeting presequence • 20 - 70 aa • 1. Many basic a.a (+ charge) = lys, arg • 2. Many hydroxylated a.a. (ser, thr) • 3. Segment can fold into a-helix + + + presequence mature protein presequence

30. Protein import into mitochondria 1) HSP70 binds & unfolds preprotein

31. Protein import into mitochondria 1) HSP70 binds & unfolds preprotein 2) Unfolded presequence binds MOM receptors (MOM19 & MOM72)

32. Protein import into mitochondria 1) HSP70 binds & unfolds preprotein 2) Unfolded presequence binds MOM receptors 3) Unfolded protein is translocated through MOM controversy: do inner and outer membrane contact each other before protein import?

33. Protein import into mitochondria 1) HSP70 binds & unfolds preprotein 2) Unfolded presequence binds MOM receptors 3) Unfolded protein is translocated through MOM 4) Unfolded protein is translocated through MIM presequence contacts MIM proteins

34. Protein import into mitochondria 5) Chaperones in matrix refold protein 2 different chaperones: mHSP70 & HSP60 consumes ATP

35. Protein import into mitochondria Driving forces for import: 1) PMF (on +ve a.a.) 2) Refolding (Brownian ratchet) 3) ATP hydrolysis used to drive unfolding and refolding

36. Protein import into mitochondria 6) Once protein is refolded, targeting sequence is removed

37. CP protein import • chaperones in cytoplasm unfold preprotein • transit peptide contacts receptor in OE • transit peptides:longer & less +ve than presequences • Just a few changes convert transit peptide to presequence

38. CP protein import • 1) chaperones in cytoplasm unfold preprotein • 2) transit peptide contacts receptor in OE • 3) Unfolded protein is translocated through OE • requires GTP • difference from mito

39. CP protein import 1) chaperones in cytoplasm unfold preprotein 2) transit peptide contacts receptor in OE 3) Unfolded protein is translocated through OE 4) Unfolded protein is translocated through IE

40. CP protein import 5) Protein is folded on inside by chaperones 6) transit peptide is removed

41. Energy for cp import 1) GTP hydolysis:crossing OE 2) Refolding (Brownian ratchet) 3) ATP hydrolysis: un- & refolding

42. CP Protein import Targeting to other parts requires another motif Hypothesis: proteins enter stroma first, then find their final destination

43. Proteomics • studying all of the proteins present in a particular organism • Now that we have the genome, what do we do with it? • old way was to prepare 2-D • gels of proteins prepared from • the cells being studied • first use isoelectric focusing • to separate proteins by pI

44. Proteomics • old way was to prepare 2-D gels of proteins prepared from the organisms or tissues being studied • first use isoelectric focusing to separate proteins by pI • Then use SDS-PAGE to separate by size

45. Proteomics • old way was to prepare 2-D gels of proteins prepared from the organisms or tissues being studied • first use isoelectric focusing to separate proteins by pI • Then use SDS-PAGE to separate by size

46. Proteomics • Use 2D-SDS-PAGE to • Separate by pI then size • Then ID each spot! • Sequencing

47. Proteomics • Use 2D-SDS-PAGE to • Separate by pI then size • Then ID each spot! • Sequencing • Slow (1aa/hr)

48. Proteomics • Use 2D-SDS-PAGE to • Separate by pI then size • Then ID each spot! • Sequencing • Slow (1aa/hr) • 98% accurate = 50 aa • limit

49. MALDI (matrix assisted laser desorption ionization) to ionize peptides so they can be analyzed by mass spectrometry. • sample is dispersed in a large excess of matrix material which absorbs the incident laser

50. sample is dispersed in a large excess of matrix material which absorbs the incident laser • Short pulses of laser light focused on the sample cause the sample and matrix to volatilize