Distinction between Endoplasmic Reticulum-Type and Plasma Membrane-Type Ca2+ Pumps (Partial Purification of a 120-Kilodalton Ca2+-ATPase from Endomembranes)

Abstract

Two biochemical types of Ca2+-pumping ATPases were distinguished in membranes that were isolated from carrot (Daucus carota) suspension-cultured cells. One type hydrolyzed GTP nearly as well as ATP, was stimulated by calmodulin, and was resistant to cyclopiazonic acid. This plasma membrane (PM)-type pump was associated with PMs and endomembranes, including vacuolar membranes and the endoplasmic reticulum (ER). Another pump (“ER-type”) that was associated mainly with the ER hydrolyzed ATP preferentially, was insensitive to calmodulin, and was inhibited partially by cyclopiazonic acid, a blocker of the animal sarcoplasmic/ER Ca2+ pump. Oxalate stimulation of Ca2+ accumulation by ER-type, but not PM-type, pump(s) indicated a separation of the two types on distinct compartments. An endomembrane 120-kD Ca2+ pump was partially purified by calmodulin-affinity chromatography. The purified polypeptide bound calmodulin reacted with antibodies to a calmodulin-stimulated Ca2+ pump from cauliflower and displayed [32P]phosphoenzyme properties that are characteristic of PM-type Ca2+ pumps. The purified ATPase corresponded to a phosphoenzyme and a 120-kD calmodulin-binding protein on endomembranes. Another PM-type pump was suggested by a 127-kD PM-associated protein that bound calmodulin. Thus, both ER- and PM-type Ca2+ pumps coexist in most plant tissues, and each type can be distinguished from another by a set of traits, even in partially purified membranes.

ECA1 complements yeast mutants defective in Ca 2+ pumps and encodes an endoplasmic reticulum-type Ca 2+ -ATPase in Arabidopsis thaliana

“…5). Thus ECA1p resembled a carrot ER-associated Ca 2ϩ pump that was partially inhibited by CPA and insensitive to calmodulin (7). Preliminary results showed that ECA1p did not bind to calmodulin.…”

“…The PM-type pump is energized by GTP or ATP and is stimulated by calmodulin. This type of pump can be located on endomembranes, like the vacuole (6, 7), as well as the plasma membrane (7,8). Another pump is associated mainly with the ER.…”

“…Another pump is associated mainly with the ER. This ER-type pump hydrolyzed ATP preferentially and was inhibited by cyclopiazonic acid (CPA), but not stimulated by calmodulin (7). Although PM-type Ca 2ϩ -ATPases have been partially purified by calmodulinaffinity chromatography, several related calmodulinstimulated Ca 2ϩ pumps could be copurified simultaneously.…”

A Novel Calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal Autoinhibitory Domain

“…Whether the plant plasma membrane-localized pumps are also members of this structurally unique subfamily is not known. However, they are reported to be larger than ACA2p (110 kDa) and are closer to the 127-136-kDa size typical of an animal PM-type Ca 2ϩ pump (5,46,49). Thus, it remains an open question whether plant cells function with two distinct types of calmodulin regulated Ca 2ϩ pumps, or operate exclusively with variants of the subfamily described here.…”

“…Thus, cells require transport systems to carefully regulate Ca 2ϩ concentrations in different cellular compartments. In plants, high affinity Ca 2ϩ -translocating ATPases are thought to function in the endoplasmic reticulum, Golgi apparatus, tonoplast, plastid inner membrane, and plasma membrane (5)(6)(7)(8). In addition, plants have low affinity Ca 2ϩ /H ϩ antiporters (9).…”