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The Protein Targeting

The Protein Targeting. Prof. V.L. Maheshwari Director, School of Life Sciences North Maharashtra University, Jalgaon. The central Dogma. DNA RNA Protein. Replication. Transcription. Reverse Transcription. Translation. Protein Biosynthesis. Major Requirements are Ribosomes

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The Protein Targeting

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  1. The Protein Targeting Prof. V.L. Maheshwari Director, School of Life Sciences North Maharashtra University, Jalgaon

  2. The central Dogma DNA RNA Protein Replication Transcription Reverse Transcription Translation

  3. Protein Biosynthesis • Major Requirements are Ribosomes Amino Acids m RNA t RNA tRNA being the translational adapter is the most important molecule. Peptide bond formation is thermodynamically unfavourable and therefore amino acids are charged

  4. cytosol nuclear envelope smooth ER lysosomes nucleus plasma membrane rough ER Proteins Golgi peroxisomes secreted mitochondria Protein sorting

  5. Protein Targetting Ribosome Free Bound Soluble Proteins Lysosomal Secretory Plasma Memb.

  6. What determines that the ribosomes will remain free or will get bound to rough ER?

  7. The signal sequence • 13-36 residues long • The N terminus always contain a positively charged amino acid • The central portion is a stretch of hydrophobic amino acids • Some proteins have internal signal sequence

  8. + + N Hydrophobic core Mature protein-C 8-12 residues cleavage site 15-20 residues Defining the signal Exceptions: not cleaved, internal signal post-translational translocation Missing elements: peptidase recognition machinery

  9. Signal Recognition Particle • Ribonucleoprotien particle, 325 kD RNA – 300 nucleotide 6 polypeptides- 9, 14, 19, 54, 68 & 78 kD 54 kD polypeptide binds to the signal sequence

  10. The SRP Receptor • Made of 2 subunits • A 69 kD alpha subunit and a 30 kD β sub unit. Alpha sub unit has positively charged amino acids. • Binding of SRP and SRP receptor is by ionic interactions.

  11. The picture so far….. ???

  12. Translocation Machinery • Multi subunit assembly of integral and peripheral membrane proteins • A few components have been identified • Protein conducting channels Gated by signal peptide 15 oA in diameter

  13. The GDP-GTP Cycle

  14. The translocation process

  15. The ER is an impressive factory • Lipid synthesis • Secretory protein synthesis • Integral membrane protein synthesis • Protein folding • Post-translational modification • Protein degradation

  16. Inside ER Lumen • Proteins are not folded immediately • Chaperon proteins keep them unfolded • Chaperons have slow ATPase activity • ADP Chaperons have high affinity for unfolded proteins • BiP (binding proteins) is a major chaperon • 78 kD hsp family protein • ER lumen also contains proteins and factors required for folding

  17. Glycosylation Core ER Glycosylation Terminal Golgi • Pentasaccharide – 3 mann and 2 GluNAc • Larger oligosaccharide is constructed on dolichol phosphate (2 GluNAc, 9 mannose and 3 glu) • Transferred to either Asn or Ser/Thr • Chaperons make sure that glucoproteins are fully folded before their export from ER

  18. The Chaperon (Bip) cycle C-ADP U Pi C-ATP U-C-ADP ATP U ADP U-C-ATP

  19. Golgi Apparatus • Major sorting centre - GPO of cell Made of 6 cisternae Cis (importing end) Medial Trans (exporting end) • Transport vesicle mediate transfer b/w ER and golgi • Small GTP binding proteins, coat proteins etc play a key role in vesicular transport

  20. Topology of eukaryotic organelles

  21. Morphology of the ER Lumen

  22. Lysosomal Targeting • Man-6 P is the marker, added in cis golgi • Added by 2 step enzyme catalysed reaction Phosphotransferase Phosphodiesterase • Man-6 P receptors in trans golgi • Fuses with pre lysosomal vesicles, acidic pH release proteins from receptors • I Cell disease- severe psychomotor retardation

  23. Protein destruction • Ubiquitin serves as a tag • It is a small 8.5 KD protein • Gets attached by its C terminal to lys of target protein • Reaction catalysed by three enzymes, E1, E2 and E3.

  24. The life of protein • Determined by N terminal amino acid • Proteins with ala, met, gly, ser, val, thr etc at the N terminus have more half life • Proteins with glu, gln, asp and asn have less half life • The tagged proteins are turned over by a 26s protease complex. • It leaves ubiquitin unaffected.

  25. Thank you

  26. Post-translational translocation requires chaperones

  27. The LDL • Major form of cholesterol transport • Contains as many as 2500 cholesterol molecules • Surrounded by a phospholipid bilayer and apoprotein B-100

  28. The LDL Receptor • Dimer of two 839 aa polypeptide • Absent in a hereditary disease called Familial Hypercholesteremia (FH)

  29. Receptor mediated endocytosis • Transport of essential metabolites (cholesterol, Vit B12, iron etc.) • Modulation of activity of protein hormones • Proteins targeted for destruction • Entry route for many viruses and toxins

  30. The process

  31. Approaches to identifying the translocon Biochemical Genetic

  32. Biochemical approach Cross link nascent proteins to the channel Stop protein in action Method of linking Method of identification

  33. Energy requirements for translocation

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