1. Post-Translational Events I Protein Trafficking
Translocation of Newly Synthesized Proteins into Membranous Organelles

2. Locations of Protein Synthesis
ER Associated Ribosomes
proteins translocate co-translationally into ER lumen
subsequent processing & transport through Golgi for secretory vesicles and lysosomes
Cytoplasmic Ribosomes
proteins translocated post-translationally into membranous organelles (except Golgi, ER and lysosome)

3. Routes of Protein Trafficking
Copyright 2008 by Saunders/Elsevier. All rights reserved.

4. Graduate students

5. Routes of Intracellular Protein Movement
Cytosolic (free) ribosomes
Cytosolic proteins
Imported into organelles
Mitochondria, Chloroplasts, Peroxisomes, Nucleus
Anchored facing cytoplasmic face of membranes
C-tail anchored proteins in mitochondrial outer membrane and ER membrane
Lipid anchored proteins on inner face of plasma membrane (via ER)
ER-associated ribosomes
Endomembrane system proteins
ER, Golgi, lysosome, vesicles, plasma membrane, secreted proteins

6. Targeting signals that direct proteins synthesized
in cytoplasm to organelles
Fig. 18-1
Copyright 2008 by Saunders/Elsevier. All rights reserved.

7. Translocons used by polypeptides to cross membranes in eukaryotes
Fig. 18-2
Copyright 2008 by Saunders/Elsevier. All rights reserved.

8. Directing Traffic Signal sequences Signal Recognition Particles
Mostly N-terminal, C-terminal
Occasionally internal
Often cleaved following transport event
Signal Recognition Particles
Bind signal sequences
Chaperone proteins
Hsp
Translocons
Protein complexes to move proteins through/into membranes

9. Trafficking of Cytosolically Synthesized Proteins
Import into organelles (transmembrane and lumenal)
Mitochondrial import
Chloroplast import
Peroxisome import
Translocation across plasma membrane
Insertion in cytoplasmic face of plasma membrane

10. Mitochondrial Protein Import
Four Destinations
Outer membrane
Innermembranous space
Inner membrane
Matrix
TOMs
Translocase of the outer mitochondrial membrane
TIMs
Translocase of the inner mitochondrial membrane
Tiny TIMs
escorts of the innermembranous space
Signal
Presequences
10-70 a.a. Basic, hydroxylate, & hydrophobic

11. Import of proteins into the mitochondrial matrix
TOM & TIM Function
Import of proteins into the mitochondrial matrix
Fig. 18-4
Copyright 2008 by Saunders/Elsevier. All rights reserved.

12. Facilitate Import into the Inner Membrane
Tiny TIMs and TIM22
Facilitate Import into the Inner Membrane
Fig. 18-5
Copyright 2008 by Saunders/Elsevier. All rights reserved.

13. TOC & TIC Import of proteins into the chloroplast
thylakoid

14. Peroxisome Import Signal sequences PTS1 Recognized by PEX5 -SKL-COOH
- RLxxxxxH/QL---

15. Peroxisome Formation

16. Translocation of ER-Associated Ribosome Synthesized Proteins

17. Endoplasmic reticulum (ER)
Fig. 20-1
Hypothesis for the evolution of the ER
Fluorescence micrograph of a cell with ER marked with a GFP fusion protein
Copyright 2008 by Saunders/Elsevier. All rights reserved.

18. Domains of the endoplasmic reticulum
Copyright 2008 by Saunders/Elsevier. All rights reserved.
Fig. 20-2

19. Docking of Ribosomes to ER

20. Signal Peptide or Signal Sequence
typically residues
shorter than those on cytosolically synthesized proteins
Hydrophobic core of ~6 residues
Examples
MKLSLVAAMLLLLSAARA BiP
MHYCVLSAFLILHLVTVAL TGF-b2
MRPSGTAGAALLALLAALCPRA EGFR

21. Signal Recognition Particle
Ribonucleoprotein complex
translocation
Ribosome binding
Signal peptide binding

22. Sec61 ER translocon

23. Structure of Sec61 translocon
Fig. 20-6
Copyright 2008 by Saunders/Elsevier. All rights reserved.

24. Targeting proteins to the ER lumen & membrane
Fig. 20-7
Copyright 2008 by Saunders/Elsevier. All rights reserved.

25. Targeting proteins to the ER membrane
Fig. 20-7
Copyright 2008 by Saunders/Elsevier. All rights reserved.

26. Translocation of Various Classes of ER Proteins
lumenal proteins
Type 1 transmembrane proteins
Type 2 transmembrane proteins
multipass transmembrane proteins

27. Post-Translational Events II
ER & Golgi Processes

28. Rough Endoplasmic Reticulum

29. Glycolsylation O-linked – serine, threonine, hydroxylysine (collagen)
N-linked - asparagine
The sugars
GalNac – N-acetylgalactosamine
GlcNac – N-acetylglucosamine
NANA – N-acetylneuraminic acid (sialic acid)
Gal – galactose
Glc – glucose
Man – mannose
Fuc – fucose
Dolicholpyrophosphoryl oligosaccharide
All sugars are linked to nucleoside mono- or diphosphate
UDP-gal, UDP-GalNac, UDP-GlcNac
GDP-man
CMP-NANA
Dolichol pyrophosphate

30. Hydroxylated amino acids

31. Glyco-nucleoside Phosphates
Dolichol phosphate
CMP-N-acetylneuraminic acid
CMP-sialic acid
UDP-galactose
UDP-N-acetylglucosamine
GDP-mannose

32. O-linked Glycosylation
Small chains of sugars
N-acetylgalactosamine (GalNac) is first sugar on S orT
GalNac transferase in ER and cis-golgi
Additional sugars added in cis, medial & trans-golgi
Gal added by galactosyl transferase
NANA added last
Each sugar addition is catalyzed by specific enzyme

33. N-linked Glycosylation
Initial step in ER
Dolichol pyrophosphoryl oligosaccharide
14 sugar moiety attached to dolichol pyrophosphate
(GalNac)2Man9Glc3
Dolichol phosphate in ER membrane
Sugars added by cytostolic glycosyltransferases
Moiety flips in ER membrane to face lumen
Transferred to asparagine by oligosaccharyl transferase (oligosaccharide-protein transferase)
Target sequence N-X-S and N-X-T (X = P)
Subsequent remodeling of sugar moiety in ER & golgi
N-linked chains very long and extensively branched

34. O- & N-linked Oligosaccharide Chains

35. N-linked Glycolsylation

36. GPI – Linkages GPI – linked proteins are attached to GPI in ER
GPI in ER membrane
C-term of protein attached to sugar residue on phosphatidyl inositol
GPI linked protein will be attached to outer leaflet of plasma membrane

37. Protein Folding Occurs in ER Lumen

38. Misfolded Proteins Reverse Translocate to Proteasome
EDEM

39. Unique Signaling from ER to Make More ER
Pathway activated by stresses that result in more unfolded proteins
heat
unfavorable pHs

40. Unfolded protein responses in the ER lumen
Fig
Activating transcription factor 6
Inositol requiring 1
PKR-like ER-kinase/pancreatic eIF2a kinase
Copyright 2008 by Saunders/Elsevier. All rights reserved.

41. Sterol & Lipid Biosynthesis
Phospholgycerides in ER
Sphingolipids in Golgi
Lipid profile asymmetry established via phospholipid exchange proteins and selective vesicular transport due to association between carrier proteins and certain lipid types

42. Regulation of HMG-CoA Reductase

43. On to the Golgi http://youtu.be/rvfvRgk0MfA