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Mariam Rizkallah - BIOT502 - Nov 15, 2011
FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from
Recycling Endosomes to the trans-Golgi Network
Mol. Biol. Cell. 2010 21(17): 3041 – 3053
Presented byMariam Rizkallah
Cell and Molecular Biology Course, The American University in Cairo
November 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Outline
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Introduction: Vesicular Traffic
Image credit: Molecular Cell Biology, Fifth Edition © 2003 W.H. Freeman and Company
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Introduction: Vesicular TrafficNascent Peptides Fate
From ER
Enzymes and structural proteins remain in ER
Transport vesicles bud to cis-Golgi
Vesicle membrane proteins recycled or missorted
bud back to ER
Cisternal maturation through Golgi
Golgi-resident proteins retrotransport to Golgi
Secretion(regulated or constitutive)
Lysosome membrane or soluble proteinsbud to late endosome
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Introduction: Where to Go?
• Soluble NSF Attachment Protein REceptor (SNAREs) crucial for cargo sorting
• Membrane budding from donor organelle, and fusion with target organelle
• Vesicle internal membrane proteins (v-SNAREs) join with target membrane proteins (t-SNAREs)
Image credit: Molecular Cell Biology, Fifth Edition © 2003 W.H. Freeman and Company
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Introduction: Where to Go?• Rab (GTPase superfamily), and Rab-mediated docking• VAMP (vesicle-associated membrane protein) as type of v-SNAREs• Syntaxin (integral) and SNAP-25 (attached) as types of t-SNAREs• SNARE coiled-coil complexes• NSF-catalyzed hydrolysis of ATP and dissociation of the SNARE complexes
Image credit: Molecular Cell Biology, Fifth Edition © 2003 W.H. Freeman and Company
Mariam Rizkallah - BIOT502 - Nov 15, 2011
The Trans-Golgi Network (TGN)
•Extension of the trans-Golgi•Sorting station (proteins and lipids)•Golgins: regulate transport and maintain structure•Cargo is either:- Transported to lysosomes (mannose 6-phosphate receptor)
- Packaged for secretion- Reside TNG (Golgins)•Problem: Which Golgin regulates what transport?
The intracellular structures known at the Golgi complex are involved in protein processing and secretion.
Image credits: Golgi - Biology Encyclopedia
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Model for FIP1/RCP Function in Retrograde Transport
Image credits: http://adoptamicrobe.blogspot.com/2006/06/escherichia-coli.html
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Retrograde Transport of TNG38, STxB and VAMP4
• Major players:- Rab11: Retrograde transport regulation- Golgin-97: TNG’s t-SNAREs (GRIP domain and FIP1/RCP-binding/coiled-coil domain)- FIP1/RCP: RE’s v-SNAREs
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Who Did It?
• Identifying the major players through:- Knockdown of Rab11a/b or its interacting protein FIP1/RCP to prove that EE/RE –and not EE/LE— to TNG transport is Rab11-mediated [Control: ]- Investigation of FIP1/RCP direct binding to Golgin-97 in vitro and in vivo to prove that binding is required for the retrograde transport from EE/RE –and not EE/LE— to the TGN [Control: CI-MPR6]
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results and Conclusions
1. Rab11 Is Required for the Localization of TGN38 to TGN
Rationale: Investigation of the role of Rab11Methods: HeLa cells-Tac-TNG38 transfected with siRNA against Rab11a and/or bKnockdown of both a/b resulted in: - Retention of TNG38
- Redistribution of FIP1/RCP to cytoplasm- Decrease in FIPs levels (FIP1/RCP and FIP5/Rip11)
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results: Rab11 Is Required for the Localization of TGN38 to TGN
Immunoblotting
Localization of FIP1/RCP
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results: Rab11 Is Required for the Localization of TGN38 to TGN
E-G: MockH-J: FIP1/RCPStaining With Antibodies
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results and Conclusions
2. FIP1/RCP Is Required for Retrograde TGN38 Transport
Rationale:Investigation of role of different FIPsMethods: HeLa cells-Tac-TNG38 transfected with siRNA against Knockdown of FIP5/Rip11, FIP1/RCP, and FIP3 resulted in:
- Redistribution of Tac-TGN38 from TGN to perinuclear punctuate organelles
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results: FIP1/RCP Is Required for Retrograde TGN38 Transport
Immunoblotting Localization of FIP1/RCP
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results: FIP1/RCP Is Required for Retrograde TGN38 Transport
C-E: MockF-H: FIP1/RCP KDI-M: Higher magnification
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Results: FIP1/RCP Is Required for Retrograde TGN38 Transport
HeLa cells treated with either mock siRNA or FIP1/RCP siRNA
Slides from 17-29 were
not prepared
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Conclusion
1. Identified Golgin-97 as binding partner for FIP1/RCP (Immunoppt and yeast two-hybrid and glutathione bead pulldown assays)
2. Knockdown results in accumulation of TGN38 in endocytic organelles containing the retromer complex (SNX1), EpsinR, and transferrin receptor.
3. Interaction between Golgin-97 and FIP1/RCP suggests that FIP1/RCP may act as a tethering factor, which mediates EE/RE-derived retrograde carrier docking and subsequent fusion at the TGN.
Mariam Rizkallah - BIOT502 - Nov 15, 2011
References
• Jing, J., Junutula, J. R., Wu, C., Burden, J., Matern, H., Peden, A. A., & Prekeris, R. (2010). FIP1/RCP binding to Golgin-97 regulates retrograde transport from recycling endosomes to the trans-Golgi network. Molecular biology of the cell, 21(17), 3041-53. doi:10.1091/mbc.E10-04-0313
• Lee, M.-S., Cherla, R. P., & Tesh, V. L. (2010). Shiga Toxins: Intracellular Trafficking to the ER Leading to Activation of Host Cell Stress Responses. Toxins, 2(6), 1515-35. Molecular Diversity Preservation International. doi:10.3390/toxins2061515
Mariam Rizkallah - BIOT502 - Nov 15, 2011
Questions
Mariam Rizkallah - BIOT502 - Nov 15, 2011
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