Heterlogous expression of a tomato plant Ca2+-ATPase in Saccharomyces cerevisiae

Jennifer Parker
Illinois State University

Several different physiological stimuli such as light, temperature, hormones, cold, and drought have been shown to elevate cytosolic [Ca2+] in plant cells. After the signaling stimuli are removed, cytosolic Ca2+ has to be returned to basal levels via active Ca2+ transport into the internal stores or the extracellular space. Plants have both high-affinity and low-affinity Ca2+-ATPase. Specifically, plants have three such transporters: high affinity plasma membrane (PM)-type Ca2+-ATPase, high affinity endoplasmic reticulum Ca2+-ATPase, and the low affinity tonoplast H+/ Ca2+ antiporter. In this study we focused on the endoplasmic reticulum Ca2+-ATPase from tomato, LCA (i.e. Lycopersicon esculentum Ca2+-ATPase).

LCA-His (LCA with an engineered six-Histidine C-terminal tag) was successfully cloned into the pYES2 vector and transformed in the Ca2+-pump devoid yeast strain, K616. Positive selection was secured on an SC-URA agar plate and LCA-His expression was induced by the inclusion 2 % galactose. Successful expression was confirmed by the observation that LCA-His complemented K616 grown in the presence of 10mM EGTA suggesting that LCA sequestered intracellular calcium. ATP-dependent 45Ca2+ transport into inside-out vesicles prepared from yeast membranes was cyclopiazonic acid (CPA)-sensitive, consistent with LCA being an ER-type Ca2+ pump. Likewise, Ca2+-dependent ATPase assays were also CPA-sensitive. Moreover, a thorough investigation of the known P-type pump inhibitors, Eosin and Vanadate, were consistent with LCA functioning as an ER-type Ca pump. Interestingly, LCA-His was shown to be thapsigargin-sensitive which contradicts previous studies of the Arabidopsis Ca2+-ATPase (ECA1), which was shown to be thapsigargin-insensitive (Liang & Sze, Plant Physiology. 118, 817-825, 1998). Investigations continue to characterize the biochemical properties of this important plant protein. Supported by a USDA grant to C.G. & Beckman Foundation fellowship to J.P.