Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans

Abstract

SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca²⁺- /Mg²⁺- dependent ATPase that sequesters Ca²⁺ into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca²⁺ homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.

Introduction

Calcium pumps, along with Ca²⁺ release channels, are essential components in maintaining intracellular Ca²⁺ homeostasis in muscle and non-muscle cells. Sequence analysis and biochemical investigation of several Ca²⁺- pumping ATPases have classified these proteins into two major classes: 1) Plasma membrane Ca²⁺-ATPase (PMCA) (Carafoli et al., 1996), and 2) Sarco (endo) plasmic reticulum Ca²⁺- ATPase (SERCA) (Wu et al., 1995, MacLennan et al., 1997). Both PMCA and SERCA have the highest affinity for Ca²⁺ mobilization from the cytoplasm and together maintain the resting cytoplasmic Ca²⁺ concentrations.

SERCA is an intracellular membrane-bound Ca²⁺-/Mg²⁺-dependent ATPase that sequesters Ca²⁺ into the SR/ER (intracellular Ca²⁺ stores) and is known to play an important role in various cellular processes (reviewed by Grover, 1985, Misquitta et al., 1999). SERCA proteins are encoded by three differentially expressed genes in mammals, namely, SERCA1, 2, and 3 (Wu et al., 1995). SERCA 1 gene products are expressed mostly in fast-twitch skeletal muscles. SERCA 2a protein is expressed at high levels in cardiac and slow-twitch striated muscles while SERCA 2b is ubiquitously expressed and, hence, is described as a housekeeping protein. SERCA 3 is alternatively spliced and produces mRNAs, encoding three protein isoforms. It is expressed in some non-muscle tissues, including intestine, thymus, cerebellum, and lymph nodes. Mutations or impaired function and expression of SERCA have shown that the protein is associated with several diseases (Just, 1996, Odermatt et al., 1996, Varadi et al., 1999, Sudbrak et al., 2000). Thus SERCA is known to occupy a central role in signal transduction, and regulate a wide range of cellular processes, including cell proliferation and apoptosis, cardiac contractility, calcium waves, and neuronal functions (see review Misquitta et al., 1999).

The nematode Caenorhabditis elegans is a popular model for developmental and genetical studies. Approximately 40% of the sequenced proteins have their homologues in other organisms (reviewed by Ahringer, 1997, The C. elegans Sequencing Consortium, 1998). A putative SERCA-type pump is found in the C. elegans genome located on chromosome III (cosmid K11D9) and is alternatively spliced to produce two isoforms. Since C. elegans represents a unique model for calcium homeostasis studies (Baylis et al., 1999, Dal Santo et al., 1999, Kraev et al., 1999, Reiner et al., 1999, Rongo and Kaplan, 1999), we undertook a functional study of the nematode SERCA. Here we report the expression pattern of C. elegans SERCA and analyzed the phenotypic effects by double-stranded RNA-mediated interference (RNAi) for the two spliced variants. Double-stranded RNAs targeted to specific isoforms resulted in embryonic lethality or L1 larval arrest, indicating that SERCA is indispensible during embryogenesis, and plays an important role in the growth and survival of C. elegans.