Mrr cmb journal_club_20111115

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


Outline


Introduction: Vesicular Traffic

Image credit: Molecular Cell Biology, Fifth Edition © 2003 W.H. Freeman and Company

http://www.youtube.com/watch?v=rvfvRgk0MfA&feature=related


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


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)



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



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


Model for FIP1/RCP Function in Retrograde Transport

Image credits: http://adoptamicrobe.blogspot.com/2006/06/escherichia-coli.html

http://adoptamicrobe.blogspot.com/2006/06/escherichia-coli.html




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


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]


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)


Results: Rab11 Is Required for the Localization of TGN38 to TGN

Immunoblotting

Localization of FIP1/RCP


Results: Rab11 Is Required for the Localization of TGN38 to TGN

E-G: MockH-J: FIP1/RCPStaining With Antibodies


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


Results: FIP1/RCP Is Required for Retrograde TGN38 Transport

Immunoblotting Localization of FIP1/RCP



C-E: MockF-H: FIP1/RCP KDI-M: Higher magnification



HeLa cells treated with either mock siRNA or FIP1/RCP siRNA

Slides from 17-29 were

not prepared














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.


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


Questions


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Mrr cmb journal_club_20111115