Manipulation of intracellular magnesium levels in Saccharomyces cerevisiae with deletion of magnesium transporters.

Magnesium is an important divalent ion for organisms. There have been a number of studies in vitro suggesting that magnesium affects enzyme activity. Surprisingly, there have been few studies to determine the cellular mechanism for magnesium regulation. We wished to determine if magnesium levels could be regulated in vivo. It is known that Saccharomyces cerevisiae has two magnesium transporters (ALR1 and ALR2) across the plasma membrane. We created S. cerevisiae strains with deletion of one (alr1 or alr2) or both (alr1 alr2) transporters. The deletion of ALR1 resulted in a decrease in intracellular magnesium levels. An increase from 5 to 100 mM in the exogenous magnesium level increased the intracellular levels of magnesium in the alr1 and alr1 alr2 strains, whereas the expression of magnesium transporters from S. cerevisiae or Arabidopsis thaliana led to a change of the intracellular levels of magnesium in those strains. The deletion of magnesium transporters in A. cerevisiae and overexpression of magnesium transporters from A. thaliana also affected the intracellular concentrations of a range of metal ions, which suggests that cells use non-specific transporters to help regulate metal homeostasis.

Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray.

Natural rubber is produced by a rubber transferase (a cis-prenyltransferase). Rubber transferase uses allylic pyrophosphate to initiate the rubber molecule and isopentenyl pyrophosphate (IPP) to form the polymer. Rubber biosynthesis also requires a divalent metal cation. Understanding how molecular weight is regulated is important because high molecular weight is required for high quality rubber. We characterized the in vitro effects of Mg(2+) on the biosynthetic rate of rubber produced by an alternative natural rubber crop, Parthenium argentatum (guayule). The affinity of the rubber transferase from P. argentatum for IPP.Mg was shown to depend on the Mg(2+) concentration in a similar fashion to the H. brasiliensis rubber transferase, although to a less extreme degree. Also, in vitro Mg(2+) concentration significantly affects rubber molecular weight of both species, but molecular weight is less sensitive to Mg(2+) concentration in P. argentatum than in H. brasiliensis.

Regulation of rubber biosynthetic rate and molecular weight in Hevea brasiliensis by metal cofactor.

Metal ion cofactors are necessary for prenyltransferase enzymes. Magnesium and manganese can be used as metal ion cofactor by rubber transferase (a cis-prenyltransferase) associated with purified rubber particles. The rubber initiation rate, biosynthetic rate, and molecular weight produced in vitro from Hevea brasiliensis rubber transferase is regulated by metal ion concentration. In addition, varies significantly with [Mg(2+)]. decreases from 8000 +/- 600 microM at [Mg(2+)] = 4 mM to 68 +/- 10 microM at [Mg(2+)] = 8 mM and increases back to 970 +/- 70 microM at [Mg(2+)] = 30 mM. The highest affinity of rubber transferase for IPP.Mg occurred when [Mg(2+)] = A(max) (metal concentration that gives highest IPP incorporation rate). A metal ion is required for rubber biosynthesis, but an excess of metal ions interacts with the rubber transferase inhibiting its activity. The results suggest that H. brasiliensis could use [Mg(2+)] as a regulatory mechanism for rubber biosynthesis and molecular weight in vivo.

Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates.

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP)