Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue

Abstract

Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo. Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not. We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release. After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger. These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.

Figure 1.

Changes in [Ca ²⁺ ] _cyt in response to hypertonic shock. (a) Luminescence response in a wild-type strain (YPH499) after addition (Time = 0) of 0.1 M CaCl₂ (gray), or after hypertonic shock treatments: 0.8 M NaCl (red); 0.9 M KCl (yellow); and 0.7 M sorbitol (blue). As measured with an osmometer, the osmolality of media with 0.8 M NaCl is the same as with 0.9 M KCl, and is ∼1.2-fold higher than with 0.7 M sorbitol. (b) Luminescence response after treatment with 0.8 M NaCl in wild-type (YPH499, black), pmc1Δ (TPYp, blue), vcx1Δ (KKY127, purple), and pmc1Δvcx1Δ (KKY124, pink) strains. All strains were transformed with PEVP11/AEQ.

Figure 2.

Phylogenetic tree of TRP family of ion channels displaying the new fungal subfamily. Ca, C. albicans; Ce, C. elegans; d, D. melanogaster; h, human; m, mouse; Nc, N. crassa; r, rat; Sc, S. cerevisiae; and TRP homologues cluster into four subfamilies.

Figure 3.

Functional characterisation of Yvc1p. (a) Yvc1–GFP localization to the vacuolar membrane, as visualized by fluorescence microscopy using an FITC filter. (b) YVC1 overexpression causes Ca²⁺ sensitivity, as shown by serial fivefold dilutions of wild-type (YPH499) strain transformed with a control (CTL) or the pYVC1-L-HA plasmid allowing high expression levels. Because YVC1-expressing cells grew slightly more slowly than control cells on regular media (unpublished data), low Ca²⁺ SD medium (see Materials and methods) was used to perform this experiment in order to have identical growth on the control plate. (c) YVC1 is required for vacuolar Ca²⁺ release in response to hypertonic shock. Luminescence response of the wild-type strain (YPH499), carrying pYVC1-U for YVC1 overexpression (WT+YVC1o.p.) or a control plasmid (WT), and of the yvc1Δ strain (VDY23) carrying a control plasmid (yvc1Δ). (d) Same experiment as c in pmc1Δvcx1Δ (KKY124) and pmc1Δvcx1Δyvc1Δ (VDY40) strains. (e) Same experiment as c in vcx1Δ (KKY127) and vcx1Δyvc1Δ (VDY31) strains.

Figure 4.

Model for vacuolar Ca ²⁺ release and sequestration. Hypertonic shock induces vacuolar Ca²⁺ release by YVC1, which requires the presence of at least one of the Ca²⁺ transporters Pmc1p or Vcx1p. After Ca²⁺ release, Vcx1p rapidly sequesters Ca²⁺ into the vacuole and decreases cytosolic [Ca²⁺].