Sterol traffic between the endoplasmic reticulum (ER) and plasma membrane (PM) is a fundamental cellular process that occurs by a poorly understood non-vesicular mechanism. We identified a novel, evolutionarily diverse family of ER membrane proteins with StART-like lipid transfer domains and studied them in yeast. StART-like domains from Ysp2p and its paralog Lam4p specifically bind sterols, and Ysp2p, Lam4p and their homologs Ysp1p and Sip3p target punctate ER-PM contact sites distinct from those occupied by known ER-PM tethers. The activity of Ysp2p, reflected in amphotericin-sensitivity assays, requires its second StART-like domain to be positioned so that it can reach across ER-PM contacts. Absence of Ysp2p, Ysp1p or Sip3p reduces the rate at which exogenously supplied sterols traffic from the PM to the ER. Our data suggest that these StART-like proteins act in trans to mediate a step in sterol exchange between the PM and ER.
