Alexander S. Zolotarev

Octopus rhodopsin Amino acid sequence deduced from cDNA

The primary structure of rhodopsin from the octopus Paroctopus defleini has been determined by parallel analysis of the protein and corresponding cDNA. The amino acid sequence is most similar to the recently cloned Drosophila opsins. Similarities to bovine and human opsins are also evident. The transmembrane topology of octopus rhodopsin is discussed.

Photosynthetic reaction centre of Chloroflexus aurantiacus. I. Primary structure of L-subunit.

The L‐subunit primary structure of the reaction centre from Chloroflexus aurantiacus composed of 310 amino acid residues has been determined by parallel analysis of the protein and corresponding DNA. Significant homology between this protein and L‐subunits from reaction centres of purple bacteria is observed. This implies close similarity in the tertiary structure of these proteins.

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

Growth and virulence of mycobacteria requires sulfur uptake. The Mycobacterium tuberculosis genome contains, in addition to the ABC sulfate permease cysTWA, three SLC26-related SulP genes of unknown function. We report that induction of Rv1739c expression in E. coli increased bacterial uptake of sulfate, but not Cl(-), formate, or oxalate. Uptake was time-dependent, maximal at pH 6.0, and exhibited a K(1/2) for sulfate of 4.0 muM. Na(+)-independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, N-ethylmaleimide and carbonyl cyanide 3-chloro-phenylhydrazone. Sulfate uptake was also increased by overexpression of the Rv1739c transmembrane domain, but not of the cytoplasmic C-terminal STAS domain. Mutation to serine of the three cysteine residues of Rv1739c did not affect magnitude, pH-dependence, or pharmacology of sulfate uptake. Expression of Rv1739c in a M. bovis BCG strain lacking the ABC sulfate permease subunit CysA could not complement sulfate auxotrophy. Moreover, inducible expression of Rv1739c in an E. coli strain lacking CysA did not increase sulfate uptake by intact cells. Our data show that facilitation of bacterial sulfate uptake by Rv1739c requires CysA and its associated sulfate permease activity, and suggest that Rv1739c may be a CysTWA-dependent sulfate transporter.

Chemical crosslinking studies with the mouse Kcc1 K–Cl cotransporter

Oligomerization, function, and regulation of unmodified mouse Kcc1 K-Cl cotransporter were studied by chemical crosslinking. Treatment of Xenopus oocytes and 293T cells expressing K-Cl cotransporter Kcc1 with several types of chemical cross-linkers shifted Kcc1 polypeptide to higher molecular weight forms. More extensive studies were performed with the amine-reactive disuccinyl suberate (DSS) and with the sulfhydryl-reactive bis-maleimidohexane (BMH). Kcc1 cross-linking was time-dependent in intact oocytes, and was independent of protein concentration in detergent lysates from oocytes or 293T cells. Kcc1 cross-linking by the cleavable cross-linker DTME was reversible. The N-terminal and C-terminal cytoplasmic tails of Kcc1 were not essential for Kcc1 crosslinking. PFO-PAGE and gel filtration revealed oligomeric states of uncrosslinked KCC1 corresponding in mobility to that of cross-linked protein. DSS and BMH each inhibited KCC1-mediated (86)Rb(+) uptake stimulated by hypotonicity or by N-ethylmaleimide (NEM) without reduction in nominal surface abundance of KCC1. These data add to evidence supporting the oligomeric state of KCC polypeptides.