Journal of Experimental Botany, Vol. 53, No. 368, pp. 565-567,
March 1, 2002
© 2002 Oxford University Press
Gene Note |
PARF-1: an Arabidopsis thaliana FYVE-domain protein displaying a novel eukaryotic domain structure and phosphoinositide affinity
Cell Signalling Group, Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Received 17 July 2001; Accepted 3 October 2001
Abstract
A full-length cDNA encoding a novel protein named PARF-1 was isolated from Arabidopsis thaliana. PARF-1 is the first eukaryotic protein to be identified that displays a domain structure which includes a FYVE-finger domain, a Pleckstrin Homology (PH) domain, as well as multiple Regulator of Chromosome Condensation-1 (RCC1) repeats. Northern blot analysis revealed that PARF-1 mRNA is present at high levels in flowers, but only at very low levels in other tissues. Recombinant PARF-1 fusion proteins expressed in E. coli were found to display unique binding specificities for monophosphorylated phosphoinositide lipids. The unusual domain structure of PARF-1 combined with its phosphoinositide specificity suggests that it may fulfil unexpected functions in higher plants.
Key words: Arabidopsis thaliana, FYVE-domain, phosphoinositides, Pleckstrin Homology (PH) domain, RCC1.
The FYVE domain is a zinc finger-like domain found in a number of eukaryotic proteins involved in membrane trafficking and signal transduction. This domain, identified by Stenmark and colleagues (Stenmark et al., 1996
), was named after the four proteins in which it was first found: Fab1, YOTB/ZK632.12, Vac1, and EEA1. The basic FYVE motif consists of eight cysteines (or in some cases seven cysteines and one histidine), two of which are part of the core motif R+HHC+XCG (where ‘+’ is a positively charged residue and ‘X’ is any amino acid). The distinguishing feature of the FYVE-domain is that it has a very high degree of specificity for the phosphoinositide lipid, phosphatidylinositol(3)phosphate, (PtdIns(3)P). PtdIns(3)P has been found in all eukaryotes, but only constitutes a minor proportion of total cellular phospholipids. The role of PtdIns(3)P in mammalian and yeast cells has been shown to be linked to aspects of vesicle trafficking and membrane sorting (Gaullier et al., 1998), but recent experiments in this laboratory suggest that PtdIns(3)P may have important additional functions in plant cells. It was shown that phosphatidylinositol 3-kinase in soybean root cells is intimately associated with active nuclear and nucleolar transcription sites (Bunney et al., 2000). One possibility is that the kinase product, PtdIns(3)P acts as a recruitment factor for proteins with potential PtdIns(3)P-binding capabilities. This prompted the search of updated Arabidopsis thaliana EST/BAC databases using BLAST algorithms against the conserved FYVE-finger domain from yeast Fab1 (Accession Code P34756). Several ESTs and a BAC from Arabidopsis thaliana which encode proteins containing putative FYVE-finger like domains were identified. One EST/BAC (EMBL accession code AC009513, and MATDB chromosome 1/BAC clone F12P19, code At1g65920) in particular attracted attention as it contained an open reading frame of 3938 nucleotides and eight introns which in addition to encoding a FYVE-domain also encodes a putative Plekstrin Homology domain (PH-domain) and a number of repeats of the mammalian Regulator of Chromosome Condensation-1 (RCC1) (accession code AAF06053).
A full length cDNA was PCR amplified from A. thaliana cDNA using the following synthetic oligonucleotide primers introducing NcoI sites at both termini to allow further cloning steps: forward 5'-CCATGGGCGAACAACAAATCTCAG-3', reverse 5'-CCCATGGCAGCATCATACTTGTTAT-3'. The PCR product was extracted from agarose gels, cloned into the pGem T-easy vector and subsequently sequenced using M13 primers and specific primers for 5' and 3' internal sequencing.
N-terminal 6-histidine tagged PARF-1–FYVE-domain fusion proteins (spanning the FYVE region shown in Fig. 2A) were expressed in E. coli strain BL21 (DE3) using the pMALc2 protein expression vector (New England Biolabs). All plasmid constructs were verified by automated DNA sequencing. His-tagged PARF-1–FYVE fusion proteins were purified from bacterial cell lysates using standard Cobalt–chelation chromatography (Clontech, Palo Alto, CA) which yielded a single protein band when analysed by SDS–PAGE.
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-helix (lower line). (B) Dot-blots of various phospholipids probed with His-tagged PARF-1–FYVE and detected by an anti-His antibody. PE, phosphatidylethanolamine; PA, phosphatidic acid; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; polyphosphoinositide names are given using IUPAC nomenclature.Total RNA was extracted from root, leaf, flower, and silique by the phenol–LiCl procedure (Verwoerd et al., 1989
). 10 µg of total RNA was separated on 5% formaldehyde+1% agarose gels, blotted onto Hybond N+ membranes (Amersham, UK) and hybridized using PARF-1 DNA probes prepared by random-primed 32P-labelling. Hybridizations were carried out at high stringency at 65 °C and washes were performed sequentially as follows: 2xSSC, 0.1% SDS for 15 min at 65 °C, 1xSSC, 0.1% SDS for 15 min at 65 °C and 0.5xSSC, 0.1% SDS for 15 min at 65 °C. Total RNA blotted onto filters was quantified by staining with methylene blue.
To assess potential PARF-1 phospholipid binding properties, protein lipid overlay experiments were performed essentially as described previously (Kavran et al., 1998; Deak et al., 1999). 2 µg aliquots of synthetic phospholipids (Echelon, Salt Lake City, UT, USA) dissolved in chloroform:methanol:water (100:100:1, by vol.) were spotted onto nitrocellulose HiBond-C extra (Amersham, UK) and allowed to dry at room temperature for 1 h. The membranes were blocked by incubation with 3% (w/v) fatty acid-free BSA in TBST (10 mM TRIS–HCl, pH 8.0, 150 mM NaCl and 0.1% (v/v) Tween-20) for 1 h at room temperature followed by overnight incubations at 40 °C with 0.5 mg ml-1 of recombinant protein solubilized in TBST+3% BSA. The membranes were washed three times for 15 min in TBST and then incubated for 1 h at room temperature with a 1/2000 dilution of an anti-His monoclonal antibody (Clontech, UK). The membranes were then washed as described above and incubated for a further 1 h with a 1/2000 dilution of anti-mouse-alkaline phosphatase antibody conjugate (Sigma, UK). After additional washes in TBST, His-tagged proteins bound to the immobilized lipids were visualized using standard procedures.
The cDNA identified in this study has 3018 nucleotides, and encodes a protein with 1006 amino acids and a molecular weight of approximately 111 kDa. This protein has been named PARF-1 due to its predicted domain structure, i.e.: PH-domain And RCC1, FYVE. The domain structure of PARF-1 is shown in Fig. 1A, and Fig. 1B shows the tissue distribution of PARF-1 mRNA. mRNA levels are high in flowers but are low, or undetectable, in all other tissues examined.
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Figure 2A
presents a more detailed comparison of the PARF-1–FYVE-domain and three canonical FYVE-domains from yeast and mammalian proteins. It can be seen that a high degree of homology exists between the Arabidopsis PARF-1–FYVE-domain and its yeast/mammalian counterparts, but the data also emphasize the important point that the PARF-1–FYVE-domain is a ‘variant-FYVE’ as the histidine and arginine in the canonical D-3 PO4-binding pocket of the FYVE-domain have been substituted by asparagine and tyrosine. As this mini-domain is highly important for PtdIns(3)P specificity (Driscoll, 2001) it is possible that PARF-1 has different specificity towards phosphoinositides than other eukaryotic proteins containing the canonical FYVE. To obtain biochemical evidence for the potential phosphoinositide-binding characteristics of the PARF-1–FYVE-domain an N-terminal 6-histidine tagged PARF-1–FYVE-domain fusion protein (spanning the FYVE region showed in Fig. 2A) was expressed in E. coli and used in dot-blot assays. Figure 2B shows dot-blots of various phospholipids probed with purified His-tagged PARF-1–FYVE. Binding was detected using an anti-His antibody and non-specific lipid–protein interactions were reduced by inclusion of 2 mM Ca2+. The data show that PARF-1–FYVE, in addition to having affinity for PtdIns(3)P, also displays a high degree of specificity for PtdIns(5)P. PARF-1–FYVE did not significantly interact with any of the other lipids tested.
The presence of the PARF-1–PH-domain further indicates the potential to interact with phosphoinositides. Although the phosphoinositide specificity of the PARF-1–PH-domain has not been investigated in detail, sequence analysis shows that it most closely resembles the PH-domain of the mammalian phospholipase C-
1 isoforms. The PLC1-PH belongs to the Rameh-classification Group II (Rameh et al., 1997) which has the highest affinity for PtdIns(3,4,5)P3 and PtdIns(4,5)P2. As PtdIns(3,4,5)P3 is currently not believed to be present in plant cells (Drøbak et al., 1999) the most likely target for the PARF-1–PH is PtdIns(4,5)P2. However, as PH-domains are known to be promiscuous with regard to phosphoinositide specificity, final conclusions must await further experimentation. The presence of both a FYVE-domain and PH-domain(s) within one protein is not unique and several eukaryotic proteins with such a domain structure have been identified. However, the presence of the additional six distinct RCC1 repeats in PARF-1 does not appear to occur in any non-plant protein so far identified. RCC1 is a nuclear chromatin-bound protein which acts as a guanine nucleotide exchange factor for the small nuclear GTPase, ran (Roepman et al., 1996), and has been found to be essential for the co-ordination of the onset of mitosis with S-phase completion in higher eukaryotic cells. RCC1 has also recently been recognized to play key roles in nucleo-cytoplasmic transport events due to its control of the guanine-nucleotide bound status of ran (Azuma and Dasso, 2000). Thus, PARF-1 has all the hallmarks of a protein with the ability to link phosphoinositide metabolism to aspects of plant nuclear function and this possibility is currently being investigated in further detail.
Acknowledgments
BH gratefully acknowledges support from EU TMR-grant ERBFMRXCT9. We thank S deVos for valuable help and JP Taylor for useful comments on the manuscript.
Notes
1 Present address: Centre for Drug Design and Development, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia. 
2 To whom correspondence should be addressed. Fax: +44 (0)1603 450022. E-mail: bjorn.drobak{at}bbsrc.ac.uk
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