No abstract

Yeast Sec14-like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high-affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Dpdr17D double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild-type. Based on the lipid profiles of wild-type and pdr16Dpdr17D cells grown in aerobic and anaerobic conditions, we suggest that PI-lanosterol transfer proteins are important predominantly for the optimal functioning of the post-lanosterol part of sterol biosynthesis.

Lipids are hydrophobic molecules with several key functions in the cell. Due to the thermodynamic constraints placed upon lipids, they cannot freely diffuse across the cytosol. Their transport between two membrane compartments can be facilitated by lipid transfer proteins (LTPs). One of the most studied families of LTPs is S. cerevisiae’s Sec14p family. Proteins belonging to this group contain cellular retinaldehyde binding – triple functional (CRAL‐TRIO) domain. In S. cerevisiae there are six members of this family, the founding member Sec14p and five of its homologues termed Sfh (Sec fourteen homologue) 1‐5. The Sec14p has been extensively studied and it was determined that it possesses phosphatidylinositol (PI) and phosphatidylcholine (PC) binding and transfer activities (Bankaitis et al., 1990). Many of Sfh proteins have been implicated in various processes in cell, however, their exact roles remain to be discovered. Lipid binding and transfer by Sfh proteins were intensively studied (Li et al., 2000; Maeda et al., 2013; Tripathi et al., 2019; Holic et al., 2014; Pevalova et al., 2019) but some key questions remain open. Our objective is to determine lipids bound to S. cerevisiae’s Sec fourteen homologue proteins. For this we used HL‐60 in situ lipid‐binding assay, which employs co‐incubation of permeabilized human HL‐60 cells with recombinant protein of interest. Afterwards, the proteins are re‐purified using affinity chromatography. The lipids extracted from re‐isolated proteins are separated using thin layer chromatography and identified. Our results showed that Sfh1 binds PC and PI with approximately the same affinity. The remaining Sfh proteins, excluding Sfh5p, predominantly bind PI and neutral lipids (NLs). Sfh5 was found to bind only PI. Analysis of bound NLs identified squalene in Sfh2p as a likely binding partner and cholesterol in association with Sfh3p and Sfh4p. Moreover, it appears that both Sfh3p and Sfh4p can bind another NL in addition to cholesterol, which remains to be identified. Based on obtained results at least three out of five Sfh proteins are capable to bind NLs, predominantly sterols or precursors of the sterol biosynthetic pathway. It appears that binding of NLs is very specific, suggesting their crucial role in the function of Sfh proteins. Thus, the activity of some members of LTP family might be regulated by sterols or their precursors which in turn can connect and synchronize the sterol biosynthetic pathway with the phospholipid trafficking events. To identify the role of sterol binding by Sfh proteins in the physiology of the yeast cell, we will attempt to prepare mutants in sterol binding. References: Bankaitis et al. (1990) Nature 347: 561Li et al. (2000) Mol Biol Cell 11: 1989Maeda et al. (2013) Nature 501: 257Tripathi et al. (2019) JBC 294:19081Holic et al. (2014) BBA 1842:1483Pevalova et al. (2019) BBA 1864: 1412