Cohen Yifat, Megyeri Márton, Chen Oscar C W, Condomitti Giuseppe, Riezman Isabelle, Loizides-Mangold Ursula, Abdul-Sada Alaa, Rimon Nitzan, Riezman Howard, Platt Frances M, Futerman Anthony H, Schuldiner Maya
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel ; Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
PLoS One. 2013 Dec 31;8(12):e85519. doi: 10.1371/journal.pone.0085519. eCollection 2013.
The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn(2+) homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn(2+) in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+) we could observe concurrent reduction in many Mn(2+)-related process in the ER lumen. Conversely, cytosolic Mn(2+)-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+) transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn(2+)-dependent neurological disorders.
内质网（ER）是一种大型、多功能且必不可少的细胞器。尽管进行了深入研究，但即使在经过充分研究的模式生物酿酒酵母中，超过三分之一的内质网蛋白的功能仍然未知。其中一种蛋白是Spf1，它是一种高度保守的、定位于内质网的假定P型ATP酶。SPF1基因的缺失会导致多种表型，包括严重的内质网应激，这表明该蛋白对于内质网的正常功能至关重要。Spf1最接近的同源物是影响锰（2+）稳态的液泡P型ATP酶Ypk9。然而，用Spf1进行的体外重组试验尚未深入了解其运输特异性。在这里，我们采用体内方法来检测缺失SPF1的直接和间接影响。我们发现，在∆spf1细胞中锰（2+）的腔浓度有特异性降低，而在其过表达后则升高。与观察到的腔内锰（2+）的损失一致，可以观察到内质网腔中许多与锰（2+）相关过程同时减少。相反，胞质中依赖锰（2+）的过程增加。总之这些数据支持Spf1p在细胞内锰（2+）运输中的作用。我们还证明了人类序列同源物ATP13A1是功能保守的直系同源物。由于ATP13A1在发育中的神经组织和大脑中高度表达这将有助于研究与锰（2+）相关的神经疾病。
