Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus.

Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes. Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner. Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L. japonicus late nodulin Nlj16. Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules. The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene. This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene. We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules.

The Schizosaccharomyces pombe spo20(+) gene encoding a homologue of Saccharomyces cerevisiae Sec14 plays an important role in forespore membrane formation.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.