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FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments

S. H. Ridley1, N. Ktistakis1, K. Davidson1, K. E. Anderson1, M. Manifava1, C. D. Ellson1, P. Lipp2, M. Bootman2, J. Coadwell1, A. Nazarian3, H. Erdjument-Bromage3, P. Tempst3, M. A. Cooper4, J. W. J. F. Thuring4, Z.-Y. Lim4, A. B. Holmes4, L. R. Stephens1 and P. T. Hawkins1,*

1 Inositide Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
2 Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
3 Memorial Sloan-Kettering Cancer Center, New York, NY10021, USA
4 Department of Chemistry, Cambridge University, Lensfield Road, Cambridge CB2 1EW, UK



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  		Fig. 1. Schematic domain profile for FENS-1 (originally catalogued as SR1; Accession Number, AJ310568) and DFCP1 (originally catalogued as SR3; Accession Number, AJ310569), also showing point mutations generated.

 


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  		Fig. 2. Analysis of phosphoinositide binding specificities of GFP-FENS-1 (A) and Myc-DFCP1 (B,C) constructs expressed in COS-7 cell lysates using lipid competition for binding to phosphoinositide-derivatised beads. The indicated GFP-tagged FENS-1 constructs or Myc-tagged DFCP1 constructs were expressed in COS-7 cells and lysates prepared as described in the Materials and Methods. Aliquots of these lysates were mixed with the indicated phosphoinositides (5 µM and 10 µM for FENS-1 and DFCP1, respectively) in a total volume of 1 ml for 10 minutes on ice, then transferred onto 15 µl packed PtdIns(3,4)P2-derivatised beads and mixed end to end at 4°C for 45 minutes. Beads were washed briefly four times and the recovery of the relevant proteins was assessed by SDS PAGE and the appropriate Western-blotting procedure (see Materials and Methods). 1% aliquots of the various lysates were taken before mixing with the beads to assess relative expression levels and recovery on the beads of each construct.

 


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  		Fig. 3. Analysis of recombinant EE-FENS-1, iFYVE FENS-1 and EE-DFCP1 binding to lipid surfaces using BiaCore. (A) Sensorgrams describing the binding of EE-FENS-1 to PC liposomes containing 1 mole% PtdIns3P or 1 mole% PtdIns5P captured on an L1 sensor chip. Binding of EE-FENS-1 to PC alone was low (approx. 20 response units at equilibrium) and this sensorgram has been subtracted from the two shown to remove bulk refractive-index changes that are due to the ethylene-glycol-containing FENS-1 solution. 100 nM FENS-1 diluted into PBS, 1 mm MgCl2 was injected at t=0 for 360 seconds (at 10 µl min-1) and then the dissociation followed for a further 400 seconds. The inset shows a Coomassie-stained SDS PAGE gel of approx. 2 µg of recombinant EE-FENS. (B) Binding of EE-FENS-1 to PC monolayers alone or containing 6 mole% phosphoinositides, assembled on an HPA sensor chip. The values shown are the means±s.e.m. (n=2-4) for the binding of approx. 100 nM EE-FENS-1 at equilibrium. (C) Sensorgram describing the binding of iFYVE FENS-1 to a PE/PC/PS (1:1:1 mole ratios) monolayer containing 1 mole% PtdIns3P assembled on an HPA sensor chip. Binding of iFYVE FENS-1 to PE/PC/PS surfaces only was low (approx 26 response units at equilibrium) and this sensorgram has been subtracted from the one shown to remove bulk refractive index changes caused by the glycerol-containing iFYVE FENS-1 solution. 100 nM iFYVE FENS-1 diluted into PBS, 1 mM MgCl2 was injected at t=0 for 360 seconds (at 10 µl min-1) and then dissociation followed for a further 400 seconds. The inset shows a Coomasie-stained SDS PAGE gel of approx 2 µg thrombin-cleaved, recombinant iFYVE FENS-1. (D) Sensorgrams describing the binding of EE-DFCP1 to PC liposomes alone or containing 1 mole% PtdIns3P or PtdIns5P, captured on an L1 sensor chip. 50 nM EE-DFCP1 diluted into PBS, 1 mM MgCl2 was injected at t=0 for 360 seconds (at 10 µl min-1) then dissociation followed for a further 400 seconds. The instant refractive index changes at the point of injection and dissociation are caused by the bulk refractive index changes to and from the ethylene glycol containing EE-DFCP1 solution. The inset shows a Coomassie-stained SDS PAGE gel of approx 2 µg EE-DFCP1.

 


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  		Fig. 4. Subcellular localisation of exogenously expressed FENS-1 and DFCP1. Top two panels: COS-7 cells were transfected with expression vectors encoding GFP-FENS-1 and GFP-DFCP1 and processed for live image recording of GFP-fluorescence as described in the Materials and Methods. The images shown represent stills taken from Movies A and B describing GFP-FENS-1 and GFP-DFCP1, respectively: the movies can be viewed at jcs.biologists.org/supplemental. Lower panel: COS-7 cells were co-transfected with expression vectors encoding GFP-FENS-1 and Myc-DFCP1. The cells were fixed with methanol and processed for direct (GFP) and indirect (Texas-Red, anti-Myc) fluorescence as described in the Materials and Methods.

 


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  		Fig. 5. Colocalisation of GFP-FENS-1 and GFP-iFYVE FENS-1 (the isolated FYVE domain) with the early endosomal marker EEA1. COS-7 cells were transfected with expression vectors encoding GFP-FENS-1 or GFP-iFYVE FENS-1. The cells were fixed with paraformaldehyde and processed for direct (GFP) and indirect immunofluorescence (Texas Red, anti-endogenous EEA1) as described in the Materials and Methods.

 


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  		Fig. 6. Effect of wortmannin on the subcellular localisation of GFP-FENS-1, GFP-iFYVE FENS-1 and GFP-DFCP1. COS-7 cells were transfected with expression vectors encoding the indicated GFP-tagged proteins and processed for live image recording of GFP-fluorescence as described in the Materials and Methods. The images shown represent stills taken at the indicated times after 100 nM wortmannin addition: the original videos describing the effects of wortmannin on the localisation of GFP-FENS-1 (Movie A) and GFP-iFYVE FENS-1 (Movie C) can be viewed at jcs.biologists.org/supplemental The cytosolic localisation of GFP-[C347S]FENS-1 is shown as an inset in the top left-hand panel.

 


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  		Fig. 7. Colocalisation of exogenously expressed DFCP1 with ER and Golgi markers. Top panels: COS-7 cells were co-transfected with expression vectors encoding GFP-elastase and Myc-DFCP1. Cells were fixed with paraformaldehyde and processed for direct (GFP) or indirect (Texas Red, anti-Myc) fluorescence as described in the Materials and Methods. The images shown were taken at high magnification with a confocal section of <1 µm on a UltraView confocal microscope. Lower panels: COS-7 cells were transfected with an expression vector encoding GFP-DFCP1. Cells were fixed with paraformaldehyde and processed for direct (GFP) or indirect (Texas-Red, anti endogenous giantin) fluorescence as described in the Materials and Methods.

 


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  		Fig. 8. Effect of exogenous expression of Myc-DFCP1 and Myc-[C647S/C770S]DFCP1 on the morphology of the Golgi. COS-7 cells were transfected with expression vectors encoding Myc-DFCP1, Myc-[C647S/C770S]-DFCP1 (DFCP1(DM)) or the Myc-tagged tandem FYVE-domains of DFCP1 (iFYVE DFCP1; see inset). Where indicated, cells were treated with 2 µg ml-1 brefeldin A (BFA) for 20 minutes before fixation. Cells were fixed with paraformaldehyde and processed for indirect immunofluorescence (fluorescein, anti-Myc; Texas-Red, anti endogenous giantin) as described in the Materials and Methods. Cells expressing Myc-DFCP1 or Myc-DFCP1(DM) are marked with an asterisk in the red, giantin images. The arrows mark a cell expressing relatively low levels of Myc-DFCP1 and consequently show good colocalisation of Myc-DFCP1 and an intact, giantin-containing compartment.

 


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  		Fig. 9. Exogenous expression of Myc-[C647S/C770S]DFCP1 (DFCP1(DM)) inhibits processing of the HA protein. COS-7 cells were co-transfected with expression vectors encoding HA and one of the indicated proteins. After lysis and immunoprecipitation, the HA was resolved in 10% SDS polyacrylamide gels, blotted and probed with HA-specific antibodies. The ER- and Golgi-forms of HA are indicated.

 

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© The Company of Biologists Ltd 2001