Protein Trafficking.pptx

This ppt about protein trafficking in cell

1. PROTEIN TRAFFICKING PRESENTED BY, NISHA GV 2 ND MS c BIOTECHNOLOGY CUSAT 1 07/02/2011

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3. Prokaryotic protein targeting The chaperone protein SecB binds to the nascent polypeptide chain to prevent premature folding which would make transport across the plasma membrane impossible SecE and SecY are transmembrane components which form a pore in the membrane through which the still unfolded polypeptide is threaded The translocation process is energy-dependent and is driven by SecA Once the protein has passed through the pore, the signal sequence is cleaved off by an extracellular, membrane-bound protease 3

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5. Two major pathways Sec and SRP Both converge at SecYEG translocon and use SecA , a peripherally bound ATPase that supplies the energy for translocation 5

6. There are two basic forms of targeting pathways: Co-translational targeting ( secretory pathway ): ER Golgi Lysosomes Plasma membrane Secreted proteins Post-translational targeting: Nucleus Mitochondria Chloroplasts Peroxisomes 6 EUKARYOTIC TRANSPORT

7. Post-translational transport is a transport of completed proteins from the cytosol into targeted organelles Organellar proteins must be targeted to the correct membrane and translocated into the organelle These proteins contain particular signal sequences that direct them to the correct organelle and compartment insertion involves signal sequence recognition by receptors on the targeted organelles 7

8. Proteins destined for secretion out of the cell are directed across the ER membrane as they are being synthesized Co-translational import Synthesis of these proteins starts but as soon as the N - terminus of the protein containing the signal sequence is made, a protein complex binds to it ( signal recognition particle) and directs the protein and ribosome to a receptor protein in the ER membrane 8

9. Peptidase cleaves the polypeptide to release the signal peptide Vesicles bud off of from the ER and Golgi, then fuse with specific organelles or the plasma membrane Vesicles bound for different destinations are coated with different types of proteins that target them to the right location 9

10. Synthesis of secretory proteins and their co translational translocation across the ER membrane 10

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12. Transport from the ER through the Golgi Apparatus The Golgi consists of a collection of flattened, membrane bound cisternae. Stack has two distinct faces: C is face (or entry face) Trans face (or exit face) Both faces are closely connected to special compartments known as the C is Golgi network (CGN) and the trans Golgi network (TGN).Between them there is Golgi stack. 12

13. 13 Proteins and lipids enter the Golgi at the CGN and they can either move onward to the TGN or be returned to the ER At the TGN they are sorted and delivered according to whether they are destined for lysosomes, secretory vesicles or the cell surface

14. Two Models for Flow of Material Through the Golgi The cisternal maturation model. As a new cis cisterna is formed it traverses the Golgi stack, changing as it matures by accumulating medial, then trans enzymes through vesicles that move from later to earlier cisternae The vesicular transport model, where each cisterna remains in one place with unchanging enzymes, and the proteins move forward through the stack via vesicles that move from earlier to later cisternae © 2006 Nature Publishing Group Malhotra , V. & Mayor, S. Cell biology: The Golgi grows up. Nature 441, 939–940 (2006) 14

15. Anterograde v s . Reterograde Transport Anterograde --- Movement of vesicles from ER through Golgi toward plasma membrane Reterograde --- Movement of vesicles from Golgi back toward the ER 15

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17. There are of three types of coated vesicles Clathrin coated COPI coated COPII coated vesicles COPI and COPII act mainly in ER or Golgi cisternae Clathrin acts in Golgi or plasma membranes 17

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19. COP proteins “cargo” More COP proteins Lipid bilayer Sar1 Cop coated vesicles contain many proteins 19

20. 1967 James Jamieson & George Palade labeled amino acids and watched movement Nobel Prize for this work(1974) in medicine to Palade 20 George Palade

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22. Import through nuclear pores Nuclear pores are large complex structures in the nuclear envelope Proteins bound for the nucleus have particular sequences of amino acids ( nuclear localization signal ) to which a nuclear import receptor protein will bind and direct the protein through the pore 22

23. Nuclear proteins like transcription factors, histones , DNA & RNA polymerases , splicing factors and ribosomal proteins are synthesized in the cytoplasm and have to be fulfill their functions Transport of proteins bigger than 20-60 kDa through NPC into the nucleus is an active process Small molecules up to 9nm in diameter can pass through NPC by passive diffusion 23

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27. Import by transport across membranes Proteins bound for the mitochondria, chloroplast, and peroxisome are make their way to the proper organelle posttranslational import The N-terminus of the protein has signal sequence which is recognized by receptor proteins in the membranes of the appropriate organelle T he protein is unfolded, then the receptor protein and a translocator assist in the transport of the polypeptide across the membrane and into the organelle O nce the signal sequence is inside, it is cleaved off by peptidase enzyme and the protein is folded into its final 3D shape ( May assistanced of molecular chaperones) 27

28. Translocation into mitochondria 28 Proteins targeted to the mitochondria are transported to the mitochondrial matrix & other targeted areas include the outer membrane, the inner membrane and the inner membrane space Mitochondrial precursor proteins bind to chaperones ( cis -acting targeting factors) in the cytosol Two chaperones have been shown to bind mitochondrial precursor proteins; these are Hsc 70 and MSF (mitochondrial import stimulation factor) These chaperones use energy released by ATP hydrolysis to keep the bound precursors in an unfolded or partially folded state. 

29. MSF has also been shown to separate protein aggregates The chaperones deliver the mitochondrial-targeted proteins to receptors located on the outer membrane of the mitochondria that are part of the TOM complex ( Translocases of the mitochondrial outer membrane) The Tom complex recognizes, binds, and translocates precursor proteins across the outer membrane of the mitochondria 29

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32. Uptake-targeting sequences of imported mitochondrial proteins 32

33. Translocation into chloroplasts T hree membranes; outer membrane, inner membrane and the thylakoid membrane Therefore the chloroplasts has three membrane spaces. The intermembranous space, the stroma and the thylakoid membrane space Proteins need to be able to go through each of the membranes and into their respected compartments Toc components mediate translocation (Toc75 is the translocon ); Hsp70/ Hsp40 may be involved 33

34. Hsp70 in both the IMS and the stroma assist the threading of the preprotein into the chloroplast An Hsp100 chaperone also called ClpC (AAA ATPase) also binds preproteins in the stroma Hsp70/ chaperonin (Cpn60) may assist folding/assembly of newly imported protein Import into thylakoid (used for respiration) uses the SRP pathway 34

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36. LYSOSOMAL TRANSPORT 36

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38. Signals in protein Organelle destination determined by signals in the amino acid sequence Particular sequences of amino acids that ultimately will bind to a receptor protein involved in importing that protein into the organelle Some cases A single sequence at the N-terminus (beginning of the protein) One or more internal sequences 38

39. SPECIFIC UPTAKE TARGETING SEQUENCES 39

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41. Protein Trafficking Diseases Over 100 inherited human diseases Defects caused by mutations in secreted proteins which prevent proper folding of the protein These mutant cargo proteins fold inefficiently and fail to exit the ER. This produces a “loss of function” phenotype Detected by a quality control system in the ER and are degraded by the ubiquitin proteasome system Restoring of trafficking has possible therapeutic strategy 41

42. Variant of the protein cystic fibrosis conductance Transmembrane regulator (CFTR), a chloride channel that controls salt balance in the lungs and pancreas. One mutation in ΔF508 lacks a phenylalanine in the first nucleotide binding domain. A defect in folding which inhibits forward transport to the Golgi. Instead, the misfolded mutant protein is targeted to the ER assisted degradation (ERAD) pathway. When the folding of ΔF508-CFTR is facilitated by incubation of cells at low temperature or by treatment of cells at physiological temperatures with pharmacological chaperones, some of the mutant protein is delivered to the plasma membrane where it displays partial chloride channel activity. FoldRx has identified compounds that stimulate this pathway 42

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45. SUMMARY ER is the first branching point In mammalian cell co translational transport occurs Polypeptide chain undergo folding within ER N-linked glycosylation in ER From protein transported to Golgi complex In Golgi protein are sorted and packed into vesicles and targeted 45

46. O-linked glycosylation in Golgi Proteins for lysosomes are phosphorylated on mannose residues Mannose 6 phosphate act as signal for lysosomes Secretory pathways involving either lysosomes or plasma membrane 46

47. REFERENCES CELLS BENJAMIN LEWIN, LYNNE CASSIMERIS, VISHWANATH R LINGAPPA, GEORGE PLOPPER THE CELL : A MOLECULAR BIOLOGY APPROACH GEOFFREY M COOPER & ROBERT E HAUSMAN MOLECULAR CELL BIOLOGY HARVEY LODISH & et al Molecular Biology of the Cell Alberts , Johnson, Lewis, Raff, Roberts & Walter 47

48. Mechanism of intracellular protein traffic.pdf Department of Biological Chemistry Protein Trafficking in the Secretory and Endocytic Pathways.pdf G817 Eukaryotic Cell Biology 2006, Simon Atkinson How Do Proteins Move Through the Golgi Apparatus? By: Pamela L. Connerly , Ph.D. (Dept .of Biology, Indiana University Southeast) © 2010 Nature Education http://web.bio.utk.edu/bcmb513 Post-translational Transport ;Targeting to the Mitochondria, Chloroplast, and Peroxisome Nucleus Protein Trafficking 48

49. http://Eukaryotic Protein Targeting/lecture8_2.html  http://bass.bio.uci.edu/7Ehudel/bs99a/lecture27/lecture8_2.html Lecture 3,Vesicular Trafficking-Cops and Clathrins-Arfs , Rabs , Sars-Snares.ppt Lecture 18: Intracellular transport.ppt Flint et al Protein Sorting & Transport.ppt PROTEIN TRAFFICKING.ppt Paul D. Brown, PhD Chapter 12:The Endomembrane System and Peroxisome.ppt http:// virtual cell animation collection PROTEIN TRAFFICKING(GOLGI). html 49

50. THANK YOU 50