A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5.

The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed "catalytic carboxylate" (E1036) and the "catalytic dyad histidine" (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.

Characterization of Thi9, a novel thiamine (Vitamin B1) transporter from Schizosaccharomyces pombe.

Thiamine is an essential component of the human diet and thiamine diphosphate-dependent enzymes play an important role in carbohydrate metabolism in all living cells. Although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe can derive thiamine from biosynthesis, both are also able to take up thiamine from external sources, leading to the down-regulation of the enzymes involved in its formation. We have isolated the S. pombe thiamine transporter Thi9 by genetic complementation of mutants defective in thiamine biosynthesis and transport. Thi9 localizes to the S. pombe cell surface and works as a high-affinity proton/thiamine symporter. The uptake of thiamine was reduced in the presence of pyrithiamine, oxythiamine, amprolium, and the thiazole part of thiamine, indicating that these compounds are substrates of Thi9. In pyrithiamine-resistant mutants, a conserved glutamate residue close to the first of the 12 transmembrane domains is exchanged by a lysine and this causes aberrant localization of the protein. Thiamine uptake is significantly increased in thiamine-deficient medium and this is associated with an increase in thi9(+) mRNA and protein levels. Upon addition of thiamine, the thi9(+) mRNA becomes undetectable within minutes, whereas the Thi9 protein appears to be stable. The protein is distantly related to transporters for amino acids, gamma-aminobutyric acid and polyamines, and not to any of the known thiamine transporters. We also found that the pyridoxine transporter Bsu1 has a marked contribution to the thiamine uptake activity of S. pombe cells.