Yu-Ping Lu

Mechanism of Heavy Metal Ion Activation of Phytochelatin (PC) Synthase BLOCKED THIOLS ARE SUFFICIENT FOR PC SYNTHASE-CATALYZED TRANSPEPTIDATION OF GLUTATHIONE AND RELATED THIOL PEPTIDES

The dependence of phytochelatin synthase (γ-glutamylcysteine dipeptidyltranspeptidase (PCS), EC 2.3.2.15) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana(AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd2+- and Zn2+-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g.Cd·GS2 or Zn·GS2) and free glutathione act as γ-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both γ-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzymes requirement for glutathione-like peptides containing blocked thiol groups for activity.

AtMRP2, an Arabidopsis ATP Binding Cassette Transporter Able to Transport Glutathione S-Conjugates and Chlorophyll Catabolites: Functional Comparisons with AtMRP1

Three ATP binding cassette (ABC) transporter–like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP–energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins.

The GS-X Pump in Plant, Yeast, and Animal Cells: Structure, Function, and Gene Expression

This review addresses the recent molecular identification of several members of the glutathione S-conjugate (GS-X) pump family, a new class of ATP-binding cassette (ABC) transporters responsible for the elimination and/or sequestration of pharmacologically and agronomically important compounds in mammalian, yeast and plant cells. The molecular structure and function of GS-X pumps encoded by MRP, cMOAT, YCF1. and AtMRP genes, have been conserved throughout molecular evolution. The physiologic function of GS-X pumps is closely related with cellular detoxification, oxidative stress, inflammation, and cancer drug resistance. Coordinated expression of GS-X pump genes, e.g., MRP1 and YCF1, and γ-glutamylcystaine synthetase, a rate-limiting enzyme of cellular glutathione (GSH) biosynthesis, has been frequently observed.

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps

Vacuolar‐type H+‐translocating pyrophosphatases (V‐PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence‐divergent V‐PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V‐PPase from Arabidopsis thaliana and the H+‐PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V‐PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M r 64 000 membrane polypeptide that specifically catalyzes Mg2+‐dependent PPi hydrolysis. The existence of PVP implies that PPi‐energized H+‐translocation is phylogenetically more deeply rooted than previously thought.