Identification of a Calmodulin-Regulated Ca2+-ATPase in the Endoplasmic Reticulum1

Abstract

A unique subfamily of calmodulin-dependent Ca 2؉ -ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca 2؉ -ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca 2؉ -ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg 2؉ , ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca 2؉ -ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals. Ca2ϩ is thought to function as an important second messenger in all eukaryotes (Bootman and Berridge, 1995;Clapham, 1995). In addition, Ca 2ϩ is required for the stability and activity of many proteins and appears to play a critical role in protein processing in the secretory pathway (Rudolph et al., 1989; Gill et al., 1996) Type IIA and IIB pumps include the "ER-type" and the "PM-type" Ca 2ϩ pumps, respectively, first characterized in animal cells. Previously, homologs of ER-or PM-type pumps were distinguished by three criteria: (a) localization to either the ER or PM, respectively, (b) differential sensitivity to inhibitors (e.g. ER-type inhibition by cyclopiazonic acid and thapsigargin), and (c) direct activation of PM-type pumps by calmodulin. However, not all plant homologs conform to these criteria (Bush, 1995;Evans and Williams, 1998).In plants several genes encoding type IIA pumps (ERtype homologs) have been cloned, including LCA1 from tomato (Wimmers et al., 1992), OsCA from rice (Chen et al., 1997), and ACA3/ECA1 (Arabidopsis Ca 2ϩ -ATPase, isoform 3/ER-Ca 2ϩ -ATPase isoform 1) from Arabidopsis (Liang et al., 1997). Consistent with the criteria for a typical ER-type pump, ACA3p (ACA isoform 3 protein) appears to reside in the ER (Liang et al., 1997). However, non-ER locations have been suggested for other isoforms. For example, Ferrol and Bennett (1996) obtained evidence for tonoplast and PM isoforms from membrane fractionation and immunodetection of pumps cross-reacting with an anti-LCA1 antibody.Three plant genes encoding type IIB pumps (PM...

Identification of a Calmodulin-Regulated Soybean Ca2+-ATPase (SCA1) That Is Located in the Plasma Membrane

“…This plasma membrane location was corroborated by cytological imaging of SCA1p tagged with a GFPp. In contrast to the fluorescent signal from a similarly tagged Ca 2ϩ pump ACA2p, which showed a reticulate-like endomembrane localization (Hong et al, 1999), SCA1-GFPp showed a peripheral pattern of fluorescence, consistent with a plasma membrane location ( Figure 5). This evidence from a GFPptagged pump was important because it provided an isoform-specific analysis coupled to a cytological verification.…”

“…of the cell, however, verified that the fluorescent signal was localized to the cell periphery, consistent with a plasma membrane location ( Figures 5B and 5C). This pattern was distinct from controls that showed non-plasma membrane locations such as a GFPp-tagged ER-localized pump (ACA2-GFPp; Hong et al, 1999) (Figure 5D) or a soluble GFPp ( Figure 5A). …”

“…First, the plant isoforms have regulatory domains located at the N terminus instead of the C terminus of the pump and show little sequence identity to the regulatory domain of an animal pump. Second, unlike type IIB pumps from animals, which are thought to be located exclusively in the plasma membrane, the plant isoforms have been found only in non-plasma membrane locations, such as ACA2p in the ER membrane (Hong et al, 1999), BCA1p in the tonoplast (Malmstrom et al, 1997), and ACA1p, which is thought to reside in the chloroplast inner membrane (Huang et al, 1993). Despite the biochemical evidence for a calmodulin-regulated Ca 2 ϩ -ATPase in the plant cell plasma membrane (Evans, 1994;Askerlund, 1997;Dainese et al, 1997;Hwang et al, 1997;Bonza et al, 1998), the corresponding gene for this has not been cloned, and it is not known whether the anticipated pump is more similar to an animal type IIB pump or to the calmodulin-regulated pumps found in plant cell endomembranes.…”

Arabidopsis Lunapark proteins are involved in ER cisternae formation

“…In addition, it also has many other functions, such as calcium homeostasis (Hong et al ., 1999), oil and protein body formation (Huang, 1996; Herman, 2008), rubber particle formation (Brown et al ., 2017) and auxin regulation (Friml & Jones, 2010; Kriechbaumer et al ., 2015). …”

Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor

“…46 Thus, Ca 2 þ homeostasis regulated by the inner membrane-localized Ca 2 þ -ATPases may have a vital role in cell death inhibitory activity of AtBI-1. Interestingly, CaM is known to bind directly to Ca 2 þ -ATPases localized at the ER, 50 suggesting a possibility that AtBI-1 may control cellular Ca 2 þ homeostasis mediated through interaction with CaM and Ca 2 þ -ATPase. Consistent with this hypothesis, an ER-resident Ca 2 þ -ATPase from tobacco (NbCA1) has recently been demonstrated to be an important modulator of PCD induced by pathogens and elicitors, with evidence that cellular Ca 2 þ fluxes are involved.…”