Regulation of CAX1, an Arabidopsis Ca(2+)/H+ antiporter. Identification of an N-terminal autoinhibitory domain

Abstract

Regulation of Ca(2+) transport determines the duration of a Ca(2+) signal, and hence, the nature of the biological response. Ca(2+)/H+ antiporters such as CAX1 (cation exchanger 1), play a key role in determining cytosolic Ca(2+) levels. Analysis of a full-length CAX1 clone suggested that the CAX1 open reading frame contains an additional 36 amino acids at the N terminus that were not found in the original clone identified by suppression of yeast (Saccharomyces cerevisiae) vacuolar Ca(2+) transport mutants. The long CAX1 (lCAX1) could not suppress the yeast Ca(2+) transport defects despite localization to the yeast vacuole. Calmodulin could not stimulate lCAX1 Ca(2+)/H+ transport in yeast; however, minor alterations in the 36-amino acid region restored Ca(2+)/H+ transport. Sequence analysis suggests that a 36-amino acid N-terminal regulatory domain may be present in all Arabidopsis CAX-like genes. Together, these results suggest a structural feature involved in regulation of Ca(2+)/H+ antiport.

Figure 1

Structure of lCAX1 compared with that of sCAX1. lCAX1 contains an additional 36 amino acids at the N terminus. This domain (shaded) has been named N1-36. The sequence of the first 37 amino acids of lCAX1 is shown. The start Met for sCAX1 is also shown. Following the identification of a sequencing error, the original nucleotide sequence of sCAX1 deposited in the GenBank database was recently amended, as previously described (Shigaki and Hirschi, 2000), thereby changing the length of the sCAX1 open reading frame from 459 amino acids to 427 amino acids. This altered open reading frame is identical to amino acids 37 to 463 of lCAX1.

Figure 2

A, Ca²⁺ tolerance assay of K667 mutant yeast-expressing vector alone (▴), sCAX1 (●), or lCAX1 (⋄). Yeast strains were grown in selection media overnight at 30°C and diluted to an optical density at 600 nm (OD₆₀₀) of 1.0, then inoculated into fresh yeast peptone dextrose (YPD) media containing a range of CaCl₂ concentrations from 100 to 200 mm. Yeast cells were grown for 16 h at 30°C in flat-bottomed 24-well dishes. Cell density was determined by measurements at OD₆₀₀. B, K667 yeast strains expressing vector alone, sCAX1, and lCAX1 were grown in selection media overnight at 30°C and diluted to an OD₆₀₀ of 1.5, then spotted onto YPD media alone and YPD media containing 200 mm CaCl₂. Yeast growth on YPD alone and YPD with 200 mm CaCl₂ was photographed after 1 and 3 d, respectively.

Figure 3

Intracellular localization of HA:sCAX1 and HA:lCAX1 in K667 mutant yeast. Yeast microsomal membranes were extracted and fractionated through a 15% to 50% (w/w) Suc gradient and 1-mL fractions were collected. Approximately 2 μg of protein from each of the seven fractions from 21% to 41% (w/w) Suc were separated by SDS-PAGE, blotted, then subjected to western-blot analyses using the anti-HA monoclonal antibody (HA:sCAX1 and HA:lCAX1) and an antibody against a yeast vacuolar membrane marker alkaline phosphatase (ALP).

Figure 4

Time course of ΔpH-dependent 10-μm ⁴⁵Ca²⁺ transport into endomembrane-enriched vesicles prepared from K667 mutant yeast expressing either A, sCAX1; B, lCAX1; or C, vector alone. Ca²⁺ transport was determined in the absence (▴) or presence (●) of 5 μm gramicidin. Ca²⁺ transport in the presence of gramicidin was not determined for the first two time points. All time course experiments were performed in the presence of 0.1 mm NaN₃, 0.2 mm Na orthovanadate, and 1 mm ATP. The Ca²⁺ ionophore A23187 (5 μm) was added at the times indicated (arrow). Results are the average (±se) of three independent experiments.

Figure 5

The ability of lCAX1 mutants with structural alterations at the N-terminal tail to suppress the Ca²⁺-sensitive growth phenotype of K667 mutant yeast. A, Schematic representation of the first 37 amino acids of lCAX1 summarizing the point mutations and truncations that were generated. Highlighted Ile residues indicate a substitution from Met. Highlighted Met residues indicate the addition of a Met that was created to initiate translation following truncation. B, Growth analysis of K667 mutant yeast expressing sCAX1, lCAX1, lCAX1-M1I, lCAX1-M37I, Δ20-lCAX1, Δ10-lCAX1, and vector alone. The yeast strains were streaked onto either plates containing YPD alone or YPD supplemented with 200 mm CaCl₂, then grown at 30°C for 2 d.

Figure 6

Time course of ΔpH-dependent 10-μm ⁴⁵Ca²⁺ transport into endomembrane-enriched vesicles prepared from K667 mutant yeast expressing either A, sCAX1; B, Δ10-lCAX1; or C, lCAX1. Ca²⁺ transport was determined in the absence (▴) or presence (●) of 5 μm gramicidin. Ca²⁺ transport in the presence of gramicidin was not determined for the first two time points. All time course experiments were performed in the presence of 0.1 mm NaN₃, 0.2 mm Na orthovanadate, and 1 mm ATP. The Ca²⁺ ionophore A23187 (5 μm) was added at the times indicated (arrow). Results are the average (±se) of two independent experiments.

Figure 7

A, Partial amino acid sequence alignment of the N-terminal tail region of various CAX-like genes from Arabidopsis (lCAX1, CAX2, and CAX3), mung bean (VCAX1), and S. cerevisiae (VCX1). The aligned sequences correspond to the entire N-terminal tails up until the first predicted transmembrane domain. The N1-36 region of lCAX1 is underlined. Alignments were performed using ClustalW 1.8 (Baylor College of Medicine Software Programs). Identical residues are shaded in black and similar residues are shaded in gray. Gaps introduced to maximize the alignment are denoted by hyphens. An asterisk denotes a putative phosphorylated residue (see B). The deduced amino acid sequence of CAX2 used here was derived from the extracted sequence of the genomic clone (accession no. AB024034). B, Amino acid sequence of the N1-36 domain of lCAX1 highlighting putative phosphorylation sites. Putative phosphorylation sites were determined using the prediction software NetPhos (Blom et al., 1999) and from analyzing known binding sites of CDPKs.