Loop 5 region is important for the activity of the long-chain base transporter Rsb1.
Intracellular lipid amounts are regulated not only by metabolism but also by efflux. Yeast Rsb1 is the only known transporter/floppase of the sphingolipid components long-chain bases (LCBs). However, even fundamental knowledge about Rsb1, such as important amino acid residues for activity and substrate recognition, still remains unclear. Rsb1 belongs to the Rta1-like family. To date, it has not been determined whether all family members share a common ability to export LCBs. Here, we revealed that within the Rta1-like family, only Rsb1 suppressed the hypersensitivity of the mutant cells lacking LCB 1-phoshate-degrading enzymes, suggesting that LCB-exporting activity is specific to Rsb1. Rsb1 contains a characteristic region (loop 5), which does not exist in other proteins of the Rta1-like family. We found that deletion of this region caused loss of Rsb1 function. Further mutational analysis of loop 5 revealed that the charged amino acid residues E223, D225 and R236 were important for Rsb1 activity. In addition to LCBs, Rsb1 facilitated the export of 1-hexadecanol, but not palmitic acid, which suggests that Rsb1 recognizes the C1 hydroxyl group. Thus, our findings provide an important clue for understanding the molecular mechanism of LCB export.
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan.
Hiroshi
H
Makuta
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan.
Keisuke
K
Obara
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan.
Akio
A
Kihara
D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
D008565:Membrane Proteins; C465340:RSB1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
lipid; long-chain base; plasma membrane; sphingolipid; transporter