Abstract
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.
Highlights
Lipids are synthesized only in specific locations in the cell and must be exported to populate membrane-bound organelles, the mechanisms of intracellular lipid traffic are still uncertain
We found a large family of eukaryotic proteins containing Steroidogenic Acute Regulatory Transfer (StART)-like domains (Figure 1 and Figure 1—figure supplement 1), which are distantly related to other domains in the StART superfamily, such as MLN64, ceramide transfer protein (CERT), Coq10p and Bet-v1 (Figure 1—figure supplement 2A)
We have shown that (i) Ysp2p is localized to puncta in the cortical ER (cER), (ii) Ysp2p can bind ergosterol via its StART-like domain, (iii) the function of Ysp2 and related genes can be partially rescued by an evolutionarily distant sterol transfer domain, and (iv) expression of Ysp2p variants can rescue the amphotericin B (AmB)-sensitivity of Δysp2 cells only when the StART-like domain is attached to a linker capable of spanning the endoplasmic reticulum (ER)-plasma membrane (PM) contact distance
Summary
Lipids are synthesized only in specific locations in the cell and must be exported to populate membrane-bound organelles, the mechanisms of intracellular lipid traffic are still uncertain. Even for organelles linked by the secretory pathway, non-vesicular mechanisms dominate for both phospholipids and sterol (Holthuis and Levine, 2005; Voelker, 2009; Holthuis and Menon, 2014). Traffic is very fast (t1⁄2 < 5 min), bidirectional, and independent of the secretory pathway (Pagano, 1990; Simons and Ikonen, 2000; Baumann et al, 2005; Mesmin et al, 2011), so non-vesicular mechanisms must exist to transfer sterol across the cytoplasm between the ER and PM. Non-vesicular sterol transport is likely to be mediated by sterol-specific lipid transfer proteins (LTPs) with the ability to extract sterols from membranes, effectively solubilizing them for transport through the cytoplasm. The dominant classes of MCS proteins are tethers (Pan et al, 2000; de Brito and Scorrano, 2008; Lahiri et al, 2014), regulators of calcium traffic (Takeshima et al, 2000; Wu et al, 2006), lipid biosynthetic enzymes
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