1. Beyond the central dogma Central dogma culminates with synthesis of protein in cytoplasm But can’t mix proteins, polysaccharides, lipids and nucleotides together and get a living cell Formation of a cell requires the context of a pre-existing cell Cell structures (organelles; mitochondria, chloroplasts, Golgi, ER) and organization must be inherited, just like DNA Epigenetics

2. Lecture 14 cont’d Intro to protein import into organelles Signal sequences  Import into the nucleus Import into mitochondria and chloroplasts Import into ER, vesicle trafficking Note - in the next few lectures I will show many figures from Molecular Biology of the Cell 4th ed. (Alberts et al.) On reserve at Marriott

3. Nuclear import occurs through pores in the double membrane All nuclear transport occurs through nuclear pores Outer nuclear membraneInner nuclear membrane “Nuclear envelope”= Nuclear lamina Perinuclear space ER ECB 15-7 Nuclear pores What molecules must be imported into nucleus? Exported?

4. Nuclear pores are large protein complexes Multiple copies of ~100 different proteins (nuclear pore proteins = NPPs) totaling >125 million daltons! Cytoplasmic face Nuclear face ECB 15-8 Cytosol Nucleus Annular subunit of central channel or transporter Nuclear basket or cage Cytplasmic fibrils Nuclear lamina Nuclear envelope

5. Transport of large molecules is active - requires GTP Small molecules (< 60 kDa), or about 9 nm diameter) enter or exit nucleus by passive diffusion Larger molecules must be actively tranported: (1)binding to transporter; and (2)transport thru nuclear pore using GTP Nuclear pores also required for active export of RNPs (including ribosome subunits, mRNA, tRNA etc.) Import and export occur through same pores

6. A nuclear localization signal (NLS) is necessary and sufficient for nuclear import of proteins The “classical” signal for nuclear import includes multiple basic amino acids (K = lysine and R = arginine)…example P-P-K-K-K-R-K-V NLS can be anywhere in protein sequence

7. Simplified view of nuclear transport (importin) NLS (cargo) ECB 15-9 Pore opens

8. GAP Molecular “switches” GTPase GTP GTPase GDPPiPi GTP “on”“off” GEF Energy for transport provided by G proteins (GTP binding proteins; large family)

9. GAP GTPase GTP GTPase GDPPiPi GDP GTP “on”“off”GEF GAP = GTPase Activating Protein GEF = Guanine Nucleotide Exchange Factor RAN GTPase used in nuclear transport

10. Q: relation to protein transport?? Nuclear import/export cycle is driven by GTP hydrolysis

11. Directional protein import is driven by GTP hydrolysis Cytoplasm Nucleus Importin (NLS receptor) binds cargo (with NLS) in cytoplasm Importin-cargo transported into nucleus thru nuclear pore Ran-GTP in nucleus binds importin, importin releases NLS (cargo) Ran-GTP-importin exported from nucleus thru pore Ran-GAP stimulates GTP hydrolysis in cytoplasm by Ran Ran-GDP releases importin in cytoplasm Ran-GDP transported into nucleus (not shown) Ran GEF stimulates nucleotide exchange restoring Ran-GTP. NLS Ran-GDP Ran-GTP Importin Ran-GTP Importin Ran-GTP Importin NLS Importin NLS Importin Ran-GDP + RanGEF GDPGTP Ran GAP PiPi

12. Specific signals direct export from the nucleus: lessons from HIV GpppCAAA mRNA (2 kB) ProcessingTranscription Unspliced vRNA (9 kB) Transport Translation Rev Cytoplasm Nucleus Human T lymphocyte HIV Reverse transcription Uncoating vRNA DS vDNATransport Integration Progeny virus exits host cell by budding Alternative splicing produces over 30 mature mRNA that are exported and translated One protein, Rev contains a NLS and is tranported into the nucleus Rev binds “Rev-response element” on vRNA Rev-RNA complex exported, RNA packaged and virus leaves cell Nuclear Export Signal Rev is req’d for export of Rev- vRNA from nucleus Human immunodeficiency virus (HIV) is a “retrovirus:” RNA genome with DS DNA intermediate RNA is “reverse transcribed” to make DS DNA Unspliced vRNA is trapped in nucleus (contains introns-no export)

13. Lecture 14 Intro to protein import into organelles Import into the nucleus  Import into mitochondria and chloroplasts

14. Organelle DNA - encodes small % proteins, human mitochondria encode only 13 proteins Rest (thousands) encoded in nucleus, transcribed, exported to cytoplasm, translated and imported into correct organelle And correct compartment in that organelle Recall mitochondrial and chloroplast structure

15. Translate mRNA for mitochondrial matrix protein in vitro Proteins contain N- terminal “signal sequence” Import into mitochondria is post-translational Digest with protease Protein degraded Add “energized” mitochondria Protein imported into mitochondria Imported matrix protein is protected from added protease Import into mitochondria and chloroplasts is post-translational Trypsin

16. Positive charge (red) clustered on one face of helix… Non-polar aa (green) on the other… Import is directed by a signal sequence at the N- terminus of mitochondrial proteins No conserved sequence Predicted to form “amphipathic” a-helix Cleaved after protein is imported MBoC (4) figure 12-23 © Garland Publishing

17. TOMs and TIMs: Import into the mitochondria matrix requires two membrane transporters… Mitochondrial import signal binds receptor in outer membrane (assoc w “TOM”) Matrix Outer membrane Inner membrane Cytoplasm Intermembrane space Matrix protein w N-terminal signal sequence Removal of signal sequence Mature matrix protein Import receptor “TOM” “TIM23” See ECB figure 15-10 “Contact site” (close apposition of OM & IM) Transport thru aqueous channels: “TOM” and “TIMs” (Translocaters in Outer/Inner Membrane)

18. Protein import into mitochondria requires energy… (1) Electrical potential (  ) across inner membrane req’d to initiate transport Adapted from MBoC (4) figure 12-27 © Garland Publishing ATP ADP + Pi - - - - - - - + + + + + + + + + + + + Cytoplasm IMS Matrix Outer membrane Inner membrane ATP ADP + Pi Cytosolic HSP70 Mitochondrial HSP70 TIM23 TOM (3) Mitochondrial HSP70 refolds protein after import (ATP used) (2) Cytosolic HSP70 unfolds protein for import (ATP used) How are proteins targeted to other mito membranes/compartments?../L14OrganelleImport/15.5-mito_import.mov

19. How are proteins targeted to mitochondrial membranes and compartments? …the direct route Cytoplasm Matrix Inner membrane Outer membrane IMS TOM TIM “Stop transfer” signal Protein in IMSProtein in IM Cleaved stop transfer (degraded) Matrix signal (cleaved and degraded) As before, signal sequence directs import through TOM/TIM23… Adapted from MBoC (4) figure 12-29 “Stop transfer” signal interrupts translocation through TIM23, releasing protein to inner membrane… Cleavage of stop transfer signal releases protein to intermembrane space…

20. Import into the thylakoid requires multiple signals A “transit peptide” (an amphipathic helix) targets to chloroplast stroma (similar to mitochondrial signal peptide, but NOT interchangeable!) Evidence for four paths to thylakoid Adapted from MBoC (4) figure 12-30 © Garland Publishing Receptor Thylakoid signal Transporters Transit peptide cleaved Transit peptide Outer membrane Inner membrane Stroma IMS Thylakoid Cytosol

21. Protein targeting Vesicle targeting Secretory vesicles Lysosomes Endosomes RetrievalTransport RER Golgi Plasma membrane See ECB figure 15-5 NLS: (basic) NES: (L-rich) Signal peptide Cytoplasm Protein targeting NucleusMitochondria Chloroplasts Additional signals for subcompartments Next two lectures

22. L15: Protein and vesicle targeting ECB 15-5 Today - import into ER, begin vesicle targeting

23. GFP-protein in plant cell ER ER network is extensive TEM of RER in dog pancreas Note ribosomes on membrane GFP-protein in plant cell ER Vesicles derived from ER by biochemical prep are termed microsomes

24. Some ribosomes bind to ER What is the evidence for cotranslational transport? ECB 15-12

25. Transport of protein into ER is cotranslational Add RER microsomes AFTER translation… Product still ~2kDa larger than in vivo product… Add protease… Product degraded… Add RER microsomes DURING translation… Product processed to mature form… Add protease… Product protected… INSIDE microsomes! In vitro product ~2 kDa larger than in vivo product ~15-25 addtnl aa at N-terminus Add protease - product degraded Translate mRNA in vitro…

26. The “Signal Hypothesis” 1.The signal for translocation of a secretory protein into the ER resides in the nascent polypeptide, in the form of a leader “pre-” sequence or “signal peptide;” 2.Translocation of the polypeptide across the ER membrane is co-translational (unlike import into nucleus, mito, and chl); and 3.the signal peptide is cleaved post-translationally in the ER lumen by a “signal peptidase.” From results of experiments such as these, Dobberstein and Blobel proposed a hypothesis Blobel - Nobel prize 1999

27. ER Signal Sequence No conserved sequence Signal sequence is 12-25 amino acids Predicted to form  -helix with hydrophobic core (yellow aa above)

28. Signal sequence is both necessary and sufficient for import into ER Necessary Sufficient

29. Requirements for targeting and translocation into the ER 1.“Signal sequence” : hydrophobic a-helix in nascent protein 2.“Signal recognition particle (SRP):” cytoplasmic complex of protein and RNA binds signal sequence 3.“SRP-receptor:” integral ER membrane protein 4.“Translocon:” an aqueous channel through ER membrane (sec61 complex)

30. Targeting to RER 1.Translation exposes signal sequence outside ribosome 2.SRP -a complex of 300bp RNA and 6 proteins- binds the signal sequence in nascent protein, transiently arrests translation 3.SRP-arrested ribosome binds SRP receptor in ER membrane (targeting) 4. Ribosome and polypeptide handed to a translocation channel (“translocon”). SRP and SRP-R are recycled (requires GTP hydrolysis). Translation resumes and translocation begins ECB 15-13

31. Proteins destined for secretion enter ER lumen Signal peptide targets nascent protein to RER as before Signal peptide is cleaved by signal peptidase associated with translocation channel Translation and translocation are completed, releasing completed polypeptide into lumen of RER Signal peptide is degraded What about membrane proteins?? ECB 15-14

32. As before, signal peptide targets nascent protein to RER However, “Stop transfer” sequence halts translocation Membrane proteins contain stop transfer sequence Protein is released from translocon Stop transfer sequence acts as transmembrane domain ECB 15-15

33. Double- and multipass membrane proteins Internal signal sequence targets nascent protein to RER… “Stop transfer” sequence halts translocation and releases protein from translocon… Signal sequence and stop transfer sequence act as transmembrane domains ECB 15-16

34. Protein folding in the ER is assisted by “BiP”… “Binding protein” (HSP70 family of ATPases) in ER lumen binds nascent polypeptide as it is being translocated, and assists folding (and translocation?)… BiP binds nascent protein during translation/translocation… RER membrane ER Lumen Translocon (sec 61 complex) Signal peptide Signal peptidase “Secreted protein” in lumen of RER Adapted from MBoC (4) figure 12-46. See ECB figure 15-14 N N C N C Signal peptide BiP ADP+Pi ATP Release of BiP from folded polypeptide requires energy (ATP)… Incorrectly folded proteins are held in ER until folded properly, or are targeted for degradation…

35. The topology of a membrane protein can be predicted… Hydrophobic  -helices of 15-25 aa are predicted to be membrane spanning domains…and also function as “topogenic sequences.” Seven domains in rhodopsin “Start transfer” initiate protein translocation, “Stop transfer” sequences halt translocation… Note start sequences can be in either orientation Adapted from MBoC (4) Figure 12-50 © Garland Publishing H 2 N--COOH 1234567 Topology of Rhodopsin COOH NH 2 ER Lumen Cytoplasm 1234567 Hydrophilic Hydrophobic 200100 Hydropathy plot for Rhodopsin Start 6 Stop 71 Start 2 Stop 3 Start 4 Stop 5 ABCD ABCD

36. Review of the “Signal Hypothesis” 1.The signal for translocation/insertion of a protein into the ER membrane resides in the nascent polypeptide, in the form of a “signal sequence.” 2.Translocation of the polypeptide across the ER membrane is co-translational 3.The signal peptide (of secreted proteins) is cleaved post-translationally in the ER lumen by a “signal peptidase.” 4.Four components: (1) signal sequence, (2) SRP, (3) SRP-R, and (4) translocon 5.Uncleaved signal sequences (and “stop transfer” sequences) function as transmembrane domains in integral membrane proteins… 6.The topology of a protein can be predicted from the “hydropathy” plot of its amino acid sequence…15.7-ERprotein_trans.mov15.7-ERprotein_trans.mov

37. Vesicle targeting

38. Protein and vesicle targeting Vesicle targeting Secretory vesicles Lysosomes Endosomes RetrievalTransport RER Golgi Plasma membrane See ECB figure 15-5 NLS: (basic) NES: (L-rich) Signal peptide Cytoplasm Protein targeting NucleusMitochondria Chloroplasts Additional signals for subcompartments… 15.1-cell_compartments.mov

39. Membrane cycling Secreted proteins Plasma membrane proteins Exocytosis (secretion) endocytosis ECB15-17 Transport is highly regulated so vesicles carry appropriate cargo for their specific destination

40. Lumen of organelle is equivalent to outside of cell x x x x x x What about membrane protein in ER? Begin with ER to Golgi

41. Modification of proteins begins in ER ECB 15-22 Disulfide bridges Glycosylation - common in plasma membrane and secreted proteins Most common glycosylation is addition of a specific oligosaccharide (14mer) to asparagine during translation. Addition is to the NH2 group; N-linked glycoproteins Addition is done in a single step by transfer from specialized dolichol lipid This oligo is then extensively modified in diverse ways Modification begins in ER: Transported to Golgi for more processing Asn-X-Ser

42. MBoC (4) figure 13-22 © Garland Publishing From the ER, proteins are transported to the Golgi Vesicular-tubular clusters to CGN RER Nuclear envelope Proteins leave the ER in transport vesicles budding from exit sites… Transport vesicles from ER fuse to form vesicular-tubular clusters… Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi network (CGN) Glycoproteins are “processed” as they pass thru the Golgi… Golgi

43. ECB 15-24 From the ER, proteins are transported to the Golgi cis-Golgi network (CGN) cis trans medial Vesicular-tubular clusters in from RER… Proteins leave the ER in transport vesicles budding from exit sites… Transport vesicles from ER fuse to form vesicular-tubular clusters… Vesicular-tubular clusters enter the Golgi by fusing with the cis-Golgi network (CGN)… Glycoproteins are “processed” as they pass thru the Golgi… Trans Golgi network (TGN)

44. The Golgi is biochemically compartmentalized… Osmium (cis) Nucleotide diphosphatase (trans) Acid phosphatase (TGN) MBoC (4) figure 13-28 © Garland Publishing

45. Glycoproteins are further processed in the Golgi GlcNAc = N-acetylglucosamine Mannose Glucose Fucose Galactose, etc. trans Plasma membrane Protein synthesis Golgi apparatus cis Secretory vesicles ERmedial Glycosylation at H 3 N + …XXNXSXX…COO - Lysosome Constituitive secretion (Default?) Regulated secretion Proteins are sorted in the TGN… Constitutive secretion… Regulated secretion… Lysosome… TGN CGN As protein moves through Golgi, monosaccharides are added or removed in specific Golgi compartments Removal of mannose Addition of GlcNAc Addition of galactose

46. Why are membrane/secreted proteins glycosylated? Structure and folding? Protection of cell (protein) from external proteases? Function? adhesion… signaling… The plasma membrane of many (most?) cells is coated with glycoproteins

47. ECB 15-24 Transport through Golgi cis-Golgi network (CGN) cis trans-Golgi network (TGN) trans medial Transport vesicles Cisternal maturation and vesicle transport probably both contribute to membrane flow through Golgi “Budding” “Fusion” 2. “Cisternal maturation” Vesicular-tubular clusters in from RER… Transport vesicles out 1. Vesicle transport

48. Next time Vesicle transport from ER to Golgi Transport from Golgi Constitutive secretion Regulated secretion To lysosome