A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca(2+)-permeable channel in the yeast vacuolar membrane.

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca(2+) activates the YVC1 current carried by cations including Ca(2+) across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

K(+)-dependent composite gating of the yeast K(+) channel, Tok1.

TOK1 encodes an outwardly rectifying K(+) channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae. It is capable of dwelling in two kinetically distinct impermeable states, a near-instantaneously activating R state and a set of related delayed activating C states (formerly called C(2) and C(1), respectively). Dwell in the R state is dependent on membrane potential and both internal and external K(+) in a manner consistent with the K(+) electrochemical potential being its determinant, where dwell in the C states is dependent on voltage and only external K(+). Whereas activation from the C states showed high temperature dependencies, typical of gating transitions in other Shaker-like channels, activation from the R state had a temperature dependence nearly as low as that of simple ionic diffusion. These findings lead us to conclude that although the C states reflect the activity of an internally oriented channel gate, the R state results from an intrinsic gating property of the channel filter region.

Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1.

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non-targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the 'post-P loop' (PP) regions. These PP mutations specifically caused marked defects in the 'C1' states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non-targeted mutagenesis and microbial selection can be a valuable tool for probing structure-function relationships of ion channels.

YKC1 encodes the depolarization-activated K+ channel in the plasma membrane of yeast.

Our previous patch-clamp studies showed that depolarization activates a K(+)-specific current in the plasma membrane of the budding yeast, Saccharomyces cerevisiae [Gustin et al. (1986) Science 233, 1195-1197]. The Yeast Genome Sequencing Project has now uncovered on the left arm of chromosome X an open reading frame (ORF) that predicts a 77-kDa protein reminiscent of a shaker-like alpha subunit with 6 membrane spans followed by a subunit with 2 spans. We found that deleting this ORF removes the yeast K+ current. Furnishing the ORF from plasmids restores or even greatly amplifies this current. These manipulations have no effects on the 40-pS mechanosensitive conductance also native to this membrane. Thus, this ORF, named YKC1 here, likely encodes a structure for the K(+)-specific channel of the yeast plasma membrane. This and other K+ channel subunits are compared and the possible uses of this gene in research are discussed. YKC1 has recently been shown by others to induce in frog oocytes a K+ current. Its activation is coupled to EK+ and its outward rectification depends on external divalent cations. We found the YKC1 channel in its native membrane activates at low voltages largely independent of EK+ and it remains so despite removal of divalents by chelation.

Reordering of nine exons is necessary to form a functional actin gene in Oxytricha nova.

During the development of a macronucleus from a micronucleus after cell mating in hypotrichs all the genes (approximately 20,000) are excised from micronuclear chromosomes as individual small DNA molecules. Telomeres are added to the ends of each gene-sized molecule and each is amplified, mostly by approximately 1000-fold, to yield a transcriptionally active macronucleus. As a part of the study of the excision of genes from chromosomes, we have cloned six fragments of chromosomal DNA from Oxytricha nova, each containing a full copy of an actin gene, for comparison with the structure of the actin-encoding DNA molecule in the macronucleus. All six micronuclear actin clones had the same overall organization as judged by restriction mapping. Two micronuclear actin clones were sequenced. These differ from one another at a few nucleotide positions but both prescribe precisely the same actin polypeptide. Both micronuclear actin genes contain nine exons separated by eight intron-like sequences. The macronuclear gene contains these nine exons without intron-like segments. Assigning the order 1 through 9 to the nine micronuclear exons, the order in the macronucleus is 8-7-1-2-4-3-5-9-6. In the micronuclear actin gene, all nine exons possess terminal repeat sequences. These repeat sequences provide precise directions for reordering and joining of the nine exons to yield the exon order in the macronuclear gene. Polymerase chain reaction analysis of micronuclear DNA of the related species, Oxytricha trifallax, shows that the actin gene has an unorthodox arrangement in this species also.

An analysis of the macronuclear actin genes of Oxytricha.

We have cloned and sequenced a 1.6-kb macronuclear molecule encoding actin from the hypotrichous ciliate Oxytricha nova. High-stringency Southern hybridization to native and digested macronuclear DNA shows that there is only one 1.6-kb actin-encoding molecule in O. nova. The 227-nucleotide 5' leader sequence contains AT-rich stretches punctuated by short GC regions. The AT-rich regions contain TATA-like sequences. However, other known eukaryote transcription regulatory sequences were not found. The 249-nucleotide 3' trailer sequence is also AT-rich and does not contain any obvious known eukaryotic mRNA processing signals. Sequence comparison with a closely related species, O. fallax, shows an 87% sequence similarity in the coding regions and an almost total lack of similarity in the noncoding regions of the molecules. However, a few small sequence similarities and motifs appear in the noncoding regions of the actin-encoding molecules of these two species. The actin-encoding molecule of O. nova could encode a polypeptide 374 amino acids long, the same size as some vertebrate cytoplasmic actins. Contrary to a previous report, we show that the actin-encoding molecule of O. fallax also codes for a polypeptide 374 amino acids long.

Expression of hepatitis B viral core region in mammalian cells.

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.