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Protein Trafficking

Protein Trafficking. Bovine cell stained with fluorescent dyes. Green -> ER Red -> Mitochondria. Major Protein sorting pathways in Eukaryotes. Secretory proteins are transported to ER shortly after synthesis started.

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Protein Trafficking

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  1. Protein Trafficking Bovine cell stained with fluorescent dyes. Green -> ER Red -> Mitochondria

  2. Major Protein sorting pathways in Eukaryotes

  3. Secretory proteins are transported to ER shortly after synthesis started

  4. Synthesis of secretory proteins and their cotranslational translocation across the ER membrane • What is needed for translocation: • Signal sequence (9-12 hydrophobic AA with some mainly pos. charged ones – in some prokaryotes sometimes longer, most of the times cleaved off by peptidases on the ER lumen side, sequence mainly at N-terminal) • Signal-Recognition-Particle (SRP) –recognizes signal sequence of ribosome complex (ribosome with mRNA), redirects ribosome complex to SRP receptor, puts synthesis of protein on hold • SRP receptor – binds the ribosome- SRP complex - driggers that ribosome complex is moved to translocon (GTP dependent) • Translocon is a protein channel, opens upon binding of ribosome complex, synthesis through channel

  5. Synthesis of secretory proteins and their cotranslational translocation across the ER membrane

  6. N-terminal signal sequence of secretary and membrane proteins

  7. Sec61α is a translocon component

  8. Translocation of secretory and membrane proteins

  9. Post-translational Translocation Fairly common in yeast and occationally in higher eukaryotes.

  10. Integral Membrane Proteins synthesized in ER

  11. Synthesis and insertion into the ER of membrane proteins Type I Type II

  12. GPI-anchored Proteins • Glycosylphosphatidylinositol (GPI) • From yeast • In other organisms -> differs in • Acyl chain • Carbohydrate moiety Formation of GPI-anchored proteins in the ER membrane

  13. Hydropathy profiles to identify topogenic sequences

  14. Protein Modification • Membrane and soluble secretary proteins synthesized on the ER have 4 possible modifications before the reach final destination: • Glycosylation in ER and Golgi • Formation of S-S bonds in ER • Proper folding and assembly of multisubunits in ER • Proteolytic cleavage in ER, Golgi, and secretory vesicles

  15. Protein Modification - Glycosylation O-linked glycosylkation: Attachment of sugars to OH of Ser and Thr Often contain only 1-4 sugar groups N-linked glycosylation: Attachment of sugars to amine N of Asn (Asn-X-Ser/Thr) Larger and more sugar groups -> more complex Glycosylation patters differ slightly between spieces !!! In Yeast: N-linked glycosylation are classified as core and mannan types. The core type contains 13-14 mannoses whereas the mannan-type structure consists of an inner core extended with an outer chain of up to 200-300 mannoses, a process known as hyperglycosylation. Precursor of N-linked sugars that are added to proteins in the ER

  16. Addition of N-linked sugars in the ER

  17. Processing of N-linked glycoproteins in the Golgi apparatus of mammalien cells Galactose addition + neuraminic acid linkage to galactose Gucosamine addition Mannose trimming

  18. Formation of S-S bond by Protein Disulfide Isomerase (PDI)

  19. Pathways for formation of S-S bonds in Eukaryotes and Bacteria

  20. Folding and assembly of Multimers Hemagglutinin trimer folding Binding of Chaperone BiP Closing S-S bond, N-linked glycosylation Membrane anchoring Assembly of trimer Another example for assembly of multimers -> immunoglobulins

  21. Improperly Folded Protein Induce Expression of Chaperons Unfolded and incomplete folded protein in the ER -> releases chaperons (BiP) from Ire1 -> upon release of BiP Ire1 dimerizes (activation) -> Endonuclease activity in th cytosol -> splices Transcription factor Hac1 -> Hac1 protein returns into nucleus -> activates transcription of Chaperons -> Misfolded and unassembled proteins -> transported from the ER to the cytosol -> degradation

  22. Modification of Proteins - Proteolytic Cleavage Proteolytic cleavage of proinsulin occurs in secretory vesicles (after Golgi)

  23. Transport of proteins to other organelles

  24. Export of Bacterial Proteins Post-translational translocation across inner membrane of gram-negative bacteria

  25. Injection of Protein by Pathogenic Bacteria (into Animal cells) Yersinia pestis: Causes Pest Virulence: Disables host macrophages -> by injecting a small set of proteins into macrophages Secretion mechanism for injecting bacterial proteins into Eukaryotic cells

  26. The secretory and endocytic pathway of protein sorting

  27. Protein Transport between Organelles are done by Vesicles Assembly of protein coat drives vesicle formation and selection of cargo molecules

  28. Assembly and Disassembly of Coat protein Interaction of cargo protein with vesicle N-terminus of Sar1 (membrane anchor) not shown

  29. Model for Docking and Fusion of Transport vesicles with Target Membrane

  30. Vesicle-mediated Protein Trafficking between ER and Golgi Backtransport mainly used for: -> recycling of membrane bilayer -> recycling of proteins (SNARE) -> missorted proteins Normal transport of secretory proteins

  31. Involvement of the 3 major types of coat proteins in traffic and secretory pathways

  32. Clathrin Coats

  33. Receptor-Mediated Endocytosis

  34. Receptor-Mediated Endocytosis

  35. Membrane Fusion directed by Hemagglutinin (HA) Influenza Virus: Glycoprotein on suface of virus After endocytosis (uptake of virus of the cell) viral envelop fuses with endosomal membrane Acidic pH necessary for conformational change in HA -> viral HA can insert into endosomal membrane

  36. HIV Budding from Plasma Membrane Gag, ESCRT and Vps4 proteins are needed ESCRT lacking -> no budding (accumulation of incomplete viral buds on membrane

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