Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant

Abstract

A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems. Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the (794)HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of delta74- At-ACA8. The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle.

The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

“…For localization purposes, a green fluorescent protein (GFP) was fused to the 3Ј ends of ACA8, ⌬74-aca8, and aca8W47A in the pYES2 vector just in front of the stop codon of the genes. ⌬74-aca8 does not encode the first 74 amino acid residues of the regulatory N terminus (19).…”

“…Both the autoinhibitory domain and the CaMBD have recently been localized to the N terminus of ACA8 (14,18,19). The CaMBD in ACA8 has been suggested to be localized between residues Ile-41 and 20).…”

“…Thus, improved ability of ACA8 mutants to complement for pmc1 and pmr1 could have resulted from re-localization of the heterologous protein within the yeast cells. Indeed, whereas ACA8 is known to be expressed in the plasma membrane of plant cells (18), Bonza et al (19) showed that ⌬74-aca8 expressed in yeast is enriched in a fraction containing membrane derived from the endoplasmic reticulum.…”

A plant plasma membrane Ca 2+ pump is required for normal pollen tube growth and fertilization

“…For complementation, a functional Ca 2ϩ pump activity is required for the secretory pathway to scavenge enough Ca 2ϩ from the cytosol for proper function. However, because ACA9 is a plasma membrane pump, its ability to complement K616 presumably resulted either from (i) a transient activity as it moved through the yeast secretory pathway or (ii) a misaccumulation of the plant pump in the yeast endoplasmic reticulum, similar to that observed for the closely related plasma membrane calcium pump ACA8 (37). In either case, complementation provides genetic evidence that ACA9 can function as a high-affinity Ca 2ϩ pump.…”

A Pollen Coat–Inducible Autoinhibited Ca2+-ATPase Expressed in Stigmatic Papilla Cells Is Required for Compatible Pollination in the Brassicaceae

“…K616 cells normally fail to grow on Ca 2+ -depleted medium. If ACA13 functions as a Ca 2+ pump, expression of ΔN-ACA13 (ACA13 lacking the 65 N-terminal amino acid residues, which comprise a predicted calmodulin-regulated autoinhibitor domain) should allow the K616 strain to grow on Ca 2+ -depleted medium, as has been demonstrated for the plasma membrane Ca 2+ pumps ACA8 and ACA9 (Bonza et al, 2004;Schiøtt et al, 2004). As shown in Figure 8, the deregulated ΔN-ACA13 indeed rescued the growth of K616 on Ca 2+ -depleted medium.…”