Abstract. We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subceUular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14 ~ defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.

BACKGROUND: This report and a companion report describe a validation of the ability of serum proteomic profiling via SELDI-TOF mass spectrometry to detect prostatic cancer. Details of this 3-stage process have been described. This report describes the development of the algorithm and results of the blinded test for stage 1.

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex. That some conservation of SEC14p function may exist was initialy suggested by experiments that revealed immuoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. We have cloned and characterized the K. lacts SEC14 gene (SEC141j). Immunoprecipitation experiments indicated that SEC14KL encoded the K. lacts structural homolog of SEC14p. In agreement with those results, nucleotide sequence analysis of SEC14x revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p. Moreover, a single ectopic copy of SEC14KL was sufficient to render S. cerevisuae secl41(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization. This efficient complementation of secl4-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis. Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well. On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported.We have been studying the Saccharomyces cerevisiae SEC14 gene product, SEC14p. SEC14p was originally recognized as being involved in yeast Golgi function by the phenotypic, biochemical, and cytological behavior exhibited by secl4(Ts) mutants upon challenge with nonpermissive conditions (16). We have subsequently shown that SEC14p most likely plays a direct role in S. cerevisiae Golgi function, represents an activity that is essential for S. cerevisiae viability, and fractionates predominantly to the S. cerevisiae cytosol (1). On the basis of such data, we have proposed that SEC14p is a cytosolic factor that stimulates an essential Golgi secretory function. In keeping with the observation that cytosolic factors involved in stimulating eucaryotic Golgi secretory function have been functionally conserved (8, 12), we discovered that the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe exhibit polypeptides that are antigenically related to the S. cerevisiae SEC14p (1). It has become a matter of of interest, therefore, to determine the precise extent of the structural similarity implicit in this immunologic cross-reactivity and to determine whether this structural similarity is indicative of functional homology as well.In this report, we present a detailed characterization of the

tsl and tS7, two temperature-sensitive mutants of Moloney murine leukemia virus strain TB induce hind-limb paralysis in 100% of CFW/D mice injected. These two paralytogenic mutants also share a defect in their inability to process the env precursor protein, Pr8Oe"v, at the restrictive temperature. To identify the mutation(s) in the genomes of the paralytogenic mutants which cause the inability to process Pr8Oe"v efficiently and confer the ability to cause hind-limb paralysis instead of lymphoma, we constructed chimeric genomes between tsl and Moloney murine leukemia virus or the TB strain of the virus. We identified a 3.9-kilobase-pair HindIII-PstI sequence from nucleotides 4895 through 8264 and 1 through 567 of tsl, comprising the 3' end of the pol and all of the env genes, the long terminal repeat, and the 5' noncoding sequence, as being responsible for the temperature sensitivity, the inefficiency in processing Pr8Oe"v, and the induction of paralysis. We extended these findings by demonstrating that the 1.6-kilobase-pair pol-gp7O HindIII-BamHI DNA sequence from nucleotides 4895 through 6537 of tsl within the 3.9-kilobase-pair HindIII-PstI fragment is necessary for tsl to induce paralysis. In addition, we showed that this 1.6-kilobase-pair fragment also controls the processing of Pr8Oe"v and the temperature sensitivity of tsl. * Corresponding author. for further investigation into the molecular mechanism of this retrovirus-induced paralysis. To identify the mutation(s) in the genomes of the paralytogenic mutants which confer temperature sensitivity, inefficient processing of Pr8Oehz', and the ability to cause hind-limb

A cDNA clone encoding a basic thaumatin-like protein of Zea mays was recovered from a mid-development seed cDNA library.l h e gene, Z/p, encoded a protein that was nearly identical with maize zeamatin and a-amylase/trypsin inhibitor. Expression of Z/p mRNA was highest in the endosperm tissue of seed 4 weeks after pollination. Expression of zeamatin-like (ZLP) protein correlated with mRNA; also, a low basal level of ZLP expression in leaf was not appreciably induced by abiotic stresses. ZLP was expressed with its own signal peptide in insect cells and in transgenic Arabidopsis and tomato plants. ZLP was secreted in all three systems, with correct processing of the signal peptide. ZLP expressed in transgenic tomato was found to be partially subjected to a proteolytic cleavage after residue 180, by an unknown mechanism, to give a "nicked" isoform of ZLP. Purified ZLP from all three sources, as well as purified "nicked" ZLP from tomato, demonstrated fungal inhibition against Candida albicans and Trichoderma reesei, with marginal inhibition observed against Alternaria solani and Neurospora crassa.