FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains.
Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His(4) and Arg(6) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn(4),Tyr(6)-->His(4),Arg(6)). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.
Jensen
R B
RB
Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, 1353 Copenhagen K, Denmark.
La Cour
T
T
Albrethsen
J
J
Nielsen
M
M
Skriver
K
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins; D017931:DNA Primers / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008081:Liposomes; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers / Q000235:genetics
D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes; D009687:Nuclear Proteins; C454959:PARF-1 protein, Arabidopsis; D018129:Phosphatidylinositol Phosphates; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C055525:phosphatidylinositol 3-phosphate; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase