Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae sec14p are found in the same cell.
Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (alpha and beta) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPalpha and PITPbeta. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.
Swigart
P
P
Department of Physiology, Rockefeller Building, 21 University Street, University College London, London WC1E 6JJ, U.K.
Insall
R
R
Wilkins
A
A
Cockcroft
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003429:Cross Reactions; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004023:Dictyostelium / Q000737:chemistry* / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D004926:Escherichia coli; D017125:Genes, Protozoan / Q000235:genetics; D005816:Genetic Complementation Test; D006056:Golgi Apparatus / Q000737:chemistry; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D049294:Phospholipid Transfer Proteins; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000378:metabolism
D002352:Carrier Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C483438:SEC14 protein, S cerevisiae; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta