Mrr cmb journal_club_20111115

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 by
Mariam Rizkallah
Cell and Molecular Biology Course,
The American University in Cairo
November 15, 2011
Mariam Rizkallah - BIOT502 - Nov 15, 2011

Outline


Introduction: Vesicular Traffic

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

Introduction: Vesicular Traffic
Nascent Peptides Fate
From ER

Enzymes and structural Transport vesicles
proteins remain in ER bud to cis-Golgi

Vesicle membrane proteins Cisternal maturation
recycled or missorted through Golgi
bud back to ER

Golgi-resident proteins Secretion Lysosome membrane
retrotransport to Golgi (regulated or soluble proteins
or constitutive) bud 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)
Image credit: Molecular Cell Biology, Fifth
Edition © 2003 W.H. Freeman and Company

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

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
The intracellular structures known at the
- Reside TNG (Golgins) Golgi complex are involved in protein
•Problem: Which Golgin regulates processing and secretion.

what transport? Image credits: Golgi - Biology Encyclopedia

Model for FIP1/RCP Function in
Retrograde Transport

Image credits: 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 Rab11
Methods:
HeLa cells-Tac-TNG38 transfected with siRNA against
Rab11a and/or b
Knockdown 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: Mock
Staining With Antibodies H-J: FIP1/RCP


Results and Conclusions
2. FIP1/RCP Is Required for Retrograde TGN38
Transport
Rationale:
Investigation of role of different FIPs
Methods:
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: Mock
F-H: FIP1/RCP KD
I-M: Higher
magnification



Slides from
17-29 were
not prepared

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


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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