From labeling studies using [14C]DCCD, the presence of two distinct DCCD‐binding sites was proved in halobacterial membrane, whose binding constants are 0.03 and 0.08 nmol DCCD/mg envelope protein, respectively. HPLC and SDS—PAGE analysis revealed that the radioactivity bound to these sites migrated with apparent molecular masses of 45 and 10.2 kDa protein components, respectively.

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) on light-induced H+-transport and transmembrane electric potential (delta phi) formation were studied in the membrane vesicles of Halobacterium halobium R1M1. In accordance with our previous finding of the existence of two DCCD-binding components in vesicle membrane using 14C-DCCD (Konishi & Murakami FEBS Lett. 169, 283-286 (1984)), DCCD inhibited the H+-influx process biphasically; that is, the H+-influx process which is electrically silent was initially inhibited at concentrations below 30 nmol of DCCD/mg vesicle protein, while another H+-influx process which is coupled to delta phi formation was secondarily inhibited above this concentration of DCCD. The latter H+-influx process was highly dependent on the Na+ concentration. The extents of Na+-dependent recovery of delta phi formation and H+-influx were quantitatively correlated. From these results, it was concluded that the second DCCD-sensitive H+-influx process which is coupled to delta phi formation is due to the hypothetical Na+/H+-antiporter postulated by Lanyi and MacDonald (Biochemistry 15, 4608-4614 (1976)). It was also found that Li+ can be substituted for Na+ in this system, as is the case with Na+/H+-antiporters found in other organisms.

No abstract

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2-3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Na+/H+ antiporter-mediated 22Na+ transport was studied in envelope vesicles from Halobacterium halobium by manipulating the size of each AfiH+ component, ApH and Av, in the dark. Neither inside alkaline ApH nor outwardly directed ApNa+, nor a combination could facilitate ZZNa+ extrusion from the vesicles. Likewise, Ay up to 144 mV (inside negative) was not capable of initiating ZZNa+ extrusion unless ApH existed. This extrusion was facilitated only when approx. 100 mV Av (gating potential) was superimposed on ApH (either 1 or 2). On the other hand, no uptake of 22Na+ took place even when both inside acidic ApH and inwardly directed Na+ gradient were imposed with or without Av. Under these conditions, monensin mediated the rapid uptake of 22Na+. The present results indicate that halobacterial Na+/H+ exchange is regulated not only by a Ayl-dependent gate but also by a certain mechanism to restrict the back flux of Na+, this making the antiporter capable of functioning as an efficient APH+-driven pump for Nat in a high saline environment. Na+/H+ antiporter; Proton electrochemical-driven Na+ pump; Membrane potential-dependent gate; (Halobacterium halobium)