We have devised a screen for genes from the yeast Saccharomyces cerevisiae whose expression is affected by cell type or by the mating pheromones. From this screen we identified a gene, FUSI, whose pattern of expression revealed interesting regulatory strategies and whose product was required for efficient cell fusion during mating. Transcription of FUSI occurred only in a and a cells, not in a/a cells, where it was repressed by al -a2, a regulatory activity present uniquely in ala cells. Transcription of FUSJ showed an absolute requirement for the products of five STE genes, STE4, STES, STE7, STEII, and STE12. Since the activators STE4, STE5, and STE12 are themselves repressed by al a2, the failure to express FUSI in a/a cells is probably the result of a cascade of regulatory activities; repression of the activators by al a2 in turn precludes transcription of FUSI. In addition to regulation of FUSI by cell type, transcription from the locus increased 10-fold or more when a or a cells were exposed to the opposing mating pheromone. To investigate the function of the Fusl protein, we created fusi null mutants. In fusi x fusl matings, the cells of a mating pair adhered tightly and appeared to form zygotes. However, the zygotes were abnormal. Within the conjugation bridge they contained a partition that prevented nuclear fusion and mixing of organelles. The predicted sequence of the Fusl protein (deduced from the FUSI DNA sequence) and subcellular fractionation studies with Fus1-pgalactosidase hybrid proteins suggest that Fusl is a membrane or secreted protein. Thus, Fusl may be located at a position within the cell where it is poised to catalyze cell wall or plasma membrane fusion.The yeast Saccharomyces cerevisiae exhibits three distinct cellular phenotypes: a, a, and a/a. The a and a cell types, which are typically haploid, can mate to yield the third cell type, an a/a diploid. These a/a diploids are not capable of mating but can be induced to undergo meiosis and sporulation, thereby regenerating haploid a and a cells (reviewed in references 18, 19, and 41). To mate efficiently, a and a cells must each secrete a specific peptide pheromone to which only the other cell type can respond. Thus, a cells secrete a 13-amino-acid-residue peptide, a factor, which binds to a specific receptor on the surface of a cells and triggers a physiological response in those cells (20,21). Similarly, a cells display a surface receptor that enables them to respond to the a-factor pheromone secreted by a cells (3,15,31). The response of a and a cells to the pheromone of the other is similar and includes an increase in the transcription of a small set of genes (16,17,43), an increase in the ability to agglutinate with the other cell type (4, 13), and arrest of the cell division cycle in the Gl phase (8,48). A mating pair whose cell cycles have been synchronized in this fashion can undergo controlled cell wall dissolution and membrane fusion to yield an a/a zygote.Despite the complexity of the cellular phenotypes outlined above, the three...

Abstract. To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOHterminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesinlike protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with transla-

Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the a mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by a cells and is transcribed only in. a cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First 1418The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.

contributed equally to this work.Receptor endocytosis is regulated by ligand binding, and receptors may signal after endocytosis in signaling endosomes. We hypothesized that signaling endosomes containing different types of receptors may be distinct from one another and have different physical characteristics. To test this hypothesis, we developed a high-resolution organelle fractionation method based on mass and density, optimized to resolve endosomes from other organelles. Three different types of receptors undergoing ligand-induced endocytosis were localized predominately in endosomes that were resolved from one another using this method. Endosomes containing activated receptor tyrosine kinases (RTKs), TrkA and EGFR, were similar to one another. Endosomes containing p75 NTR (in the tumor necrosis receptor superfamily) and PAC1 (a G-protein-coupled receptor) were distinct from each other and from RTK endosomes. Receptorspecific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.