Molecular characterization of Rab11-FIP3 binding to ARF GTPases.
Rab11-FIP3 is a Rab11-binding protein that has been implicated in regulating cytokinesis in mammalian cells. FIP3 functions by simultaneously interacting with Rab11 as well as Arf GTPases. However, unlike the interaction between Rab11 and FIP3, the structural basis of FIP3 binding to Arf GTPases has not yet been determined. The specificity of interaction between FIP3 and Arf GTPases remains controversial. While it was reported that FIP3 preferentially binds to Arf6 some data suggest that FIP3 can also interact with Arf5 and even possibly Arf4. The Arf-interaction motif on FIP3 also remains to be determined. Finally, the importance of Arf binding to FIP3 in regulating cell division and other cellular functions remains unclear. Here we used a combination of various biochemical techniques to measure the affinity of FIP3 binding to various Arfs and to demonstrate that FIP3 predominantly interacts with Arf6 in vitro and in vivo. In addition, we identified the motifs mediating Arf6 and FIP3 interaction and demonstrated that FIP3 binds to the Arf6 C-terminus rather than switch motifs. Finally we show that FIP3 and Arf6 binding is required for the targeting of Arf6 to the cleavage furrow during cytokinesis. Thus, we propose that FIP3 is a scaffolding protein that, in addition to regulating endosome targeting to the cleavage furrow, also is required for Arf6 recruitment to the midbody during late telophase.
Schonteich
Eric
E
Department of Cellular and Developmental Biology, School of Medicine, University of Colorado Health Sciences Center, 12801 E. 17th Avenue, Aurora, CO 80045, USA.
Pilli
Manohar
M
Simon
Glenn C
GC
Matern
Hugo T
HT
Junutula
Jagath R
JR
Sentz
Danielle
D
Holmes
Randall K
RK
Prekeris
Rytis
R
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D048749:Cytokinesis; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000089982:ADP-Ribosylation Factor 6; D002352:Carrier Proteins; D050505:Mutant Proteins; C439063:RAB11FIP3 protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human