How does a protein get to the correct cellular location?

1. How does a protein get to the correct cellular location? • Membrane and organelle proteins contain targeting (sorting) signals in their amino acid sequence. • Targeting signals are recognized during or after the protein is translated - special machinery recognizes the signal and translocates the protein to its correct location

2. Examples of protein targeting signals

3. Proteins are targeted to different compartments in different ways

4. Proteins that are targeted to the nucleus, mitochondria, chloroplasts and peroxisomes are synthesized on free ribosomes as soluble polypeptides

5. Proteins that are targeted to the cell surface, Golgi and Lysosomes are synthesized on ER membrane bound ribosomes and move through the secretory pathway

6. Overview of secretory pathway

7. All proteins encoded by nuclear DNA are first translated on free cytoplasmic ribosomes • Soluble proteins and proteins targeted to the mitochondria, chloroplasts and peroxisomes are completely synthesized on free ribosomes • Translation of Integral membrane proteins, secreted proteins, and proteins in the ER, Golgi, and lysosomes are synthesized on ribosomes bound to the ER membrane • The subunits on free and ER bound ribosomes are identical

8. What happens when protein targeting doesn’t work? • I-cell disease caused by defect in lysosomal targeting • Many hydrolytic enzymes fail to be targeted to lysosomes and are secreted from cells • Psychomotor retardation, skeletal abnormalities • Average lifespan ~ 8 years

9. Zellwenger syndrome • Peroxisomal targeting defect • Peroxisomal enzymes accumulate in cytosol • Neural, cardiovascular, renal, adrenal dystrophies • Accumulate very long chain fatty acids • Cataracts, glaucoma, retinal detachment • Average lifespan - 12 weeks

10. Protein Targeting:How do secreted proteins get to the ER membrane? Gunther BlobelNobel Prize 1999"for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell"

11. Early experiments: An N-terminal signal sequence on nascent secretory proteins targets synthesis to the ER and is then cleaved. Translation of secretory mRNA’s in cell free protein synthesis system produces full length proteins with intact signal sequence Adding microsomes (ER membranes) to system causes ribosomes to bind to membranes, translocation of the protein to the lumen and cleavage of signal sequence

12. Signal sequences usually contain 1 or more + charged amino acids followed by a stretch of hydrophobic residues

13. The ER targeting mechanism requires two special receptor proteins: What gets the ribosomes with secretory protein mRNA's to bind to the ER membranes? 1. Signal recognition particle (SRP) 2. SRP receptor

14. Translation of secretory mRNA begins on free ribosomes • N-terminal signal sequence emerges from ribosome tunnel • Signal recognition particle (SRP) binds to the emerging signal sequence from the ribosome

15. SRP is a ribonucleoprotein • 300 base RNA molecule • 6 proteins • Methionine "whiskers" on P54 subunit bind to the hydrophobic signal sequence on the emerging polypeptide

16. Cryo-EM map SRP and 80S ribosomeNature427, 808 - 814 (26 February 2004

17. SRP –ribosome interaction EFS: elongation factor binding site

18. Exam study recommendation Some questions on exam are to test the understanding of functions. e.g. “What would be the effect of a loss of function mutation in the signal binding part of the Signal Recognition particle?”

19. SRP receptor initiates the interaction of signal sequences with the ER membrane • Receptor is an a,b dimer – b subunit is an intrinsic membrane protein • a-subunit initiates binding of ribosome –SRP to ER membrane

20. SRP/SRP receptor dissociates from signal sequence • Ribosome binds to translocon • Signal sequence binds to translocon. Translocon gate opens • Signal sequence inserts into translocon central cavity w/ N-terminus toward cytosol

22. Polypeptide chain elongates; signal sequence cleaved and degraded in ER lumen • Peptide chain elongation extrudes protein into ER lumen

23. Sec63 complex promotes Hsc70 chaperone (BiP) binding to growing chain

24. Ribosome dissociates and is released from membrane when protein is completed

25. What controls the insertion of nascent secretory proteins into the translocon? • The P54 subunit of the Signal Recognition Particle is a GTPase • So is the a-subunit of the SRP receptor GTP binding to both proteins produces conformational changes required for tight “docking” to the membrane

26. GTP hydrolysis initiates protein transport into the ER

27. GTP hydrolysis powers 1) dissociation of SRP, SRP receptor from translocon, 2) opening of translocon gate, 3) transfer of signal sequence to translocon

28. SRP and SR stimulate each other's GTPase activity.GTP hydrolysis triggers unidirectional targeting of ribosome/cargo binding to the Sec61α translocation pore.

29. What other GTP hydrolysis mechanisms power protein translocation into the ER?

30. Peptide bond formation

31. Secretory proteins move from the Rough ER lumen through Golgi complex and then to cell surface by vesicle mediated transport This is driven by energy released during protein translation

32. How do intrinsic membrane proteins get inserted into the ER membrane?

33. Topologies of some integral membrane proteins synthesized on the rough ER

34. Most cytosolic transmembrane proteins have an N-terminal signal sequence and an internal topogenic sequence Type I protein

35. A single internal signal-anchor sequence directs insertion of single-pass Type II transmembrane proteins Type II protein, no N-terminal signal sequence

36. Multipass transmembrane proteins have multiple topogenic sequences

37. After insertion into the ER membrane, some proteins are transferred to a GPI anchor

39. Post-translational modifications and quality control in the rough ER • Newly synthesized polypeptides in the membrane and lumen of the ER undergo five principal modifications • Formation of disulfide bonds • Proper folding • Addition and processing of carbohydrates • Specific proteolytic cleavages • Assembly into multimeric proteins

40. Disulfide bonds are formed and rearranged in the ER lumen

41. Most proteins synthesized in the Rough ER are glycosylated by a core oligosaccharide that is linked to asparagine residues(N-linked glycosylation)

42. The glycosylation signal is Asn-X- (Ser/Thr)

43. The core oligosaccharide used for N-linked glycosylation is assembled onto the polyisoprenoid lipid, dolichol pyrophosphate

44. Dolichol is an poly - isoprenoid compound synthesized by the same metabolic route as cholesterol. • In vertebrate tissues, dolichol contains 18-20 isoprenoid units (90-100 carbons total).

45. Formation of the Core Oligosaccharide on Dolichol Phosphate starts in the cytosol and is completed in the ER lumen

46. N-linked glycosylation occurs during protein translocation via the membrane bound protein oligosaccharide transferase

47. Core Glycosylation and Trimming in the ER lumen

48. Oligosaccharide Transferase Glucose and Mannose Trimming

49. Correct folding of newly made proteins is facilitated by several ER proteins that bind to oligosaccharides • Calnexin and Calreticulin are Ca++ binding proteins that bind to glucosylated oligosaccharides of incompletely folded proteins • promote association with protein disulfide isomerase which facilitates formation of correct disulfide bonds • prevent incompletely folded proteins from irreversible aggregation before disufide bond formation and intitial folding occurs

50. Bip, Calnexin and Calreticulin binding promote folding of adjacent areas while correct disulfide bonds are formed