Tritium suicide selection has been used successfully to isolate bacterial and yeast mutants with defects in lipid biosynthesis, protein N-glycosylation, and vesicular transport (8
). We used this approach to identify genes involved in the uptake and intracellular transport of sterols. Both cholesterol and ergosterol are efficiently taken up by Upc2-1
cells. Exogenously supplied ergosterol equilibrates with endogenous ergosterol in the PM and is consequently found mostly in the free (nonesterified) form in cells (32
). Cholesterol partitions less well into the PM, possibly because it is excluded from privileged interactions with yeast sphingolipids (or other lipids with saturated acyl chains) when ergosterol is present. Consequently, exogenously supplied cholesterol transits through the PM and moves to the ER, where it is esterified by the acyl-coenzyme A cholesterol acyl transferase enzymes Are1 and Are2. We exploited this transport-dependent accumulation of exogenous cholesterol to perform [3
H]cholesterol suicide selection on Upc2-1
cells mutagenized with a transposon library. We expected to recover mutants with defects in the uptake, transport, and esterification processes. From the survivors of the selection, 23 genes were identified; disruption of these genes by transposon insertion resulted in a defect in cholesterol uptake. Numerous genes were isolated only once (Table ), indicating that coverage of the library was incomplete and that more mutants would likely be uncovered by extending or repeating the selection process. Of the genes we identified, HOF1
and genes encoding components of the Ras signaling pathway (RAS2
) were also identified in a recent genomewide screen for genes involved in neutral lipid storage (11
). Although the role of these gene products in sterol homeostasis is unclear, their repeated isolation not only validates both approaches—the published genomewide screen (11
) and our suicide selection—for identifying new mutants but also confirms the importance of these proteins in intracellular sterol regulation. Interestingly, neither approach yielded mutants of OSH3
, which would be expected to display a reduced rate of cholesterol accumulation (39
). Also, none of the genes that we identified were uncovered in a screen of anaerobic inviable mutants (40
), even though this screen found numerous genes involved in the trafficking of Aus1 and Pdr11 to the PM. This lack of overlap is likely due to the incomplete coverage of the genome in our suicide selection process; by continuing the selection process, we would undoubtedly uncover a number of the reported anaerobic inviable mutants as well as mutants with transposon insertions in OSH3
Figure envisages three steps in the accumulation of exogenously supplied cholesterol. C209 (det1::mTn3) cells esterify cholesterol similarly to parental Upc2-1 cells and display a normal pattern of expression of Aus1 and Pdr11 (Fig. ), suggesting that the defect in these cells is in the step of sterol transport between the ER and PM rather than in sterol uptake at the PM or sterol esterification. In support of this, assays of biosynthetic sterol transport in det1Δ cells revealed that transport of newly synthesized ergosterol from the ER to the PM was slowed threefold compared to that in parental BY4741 cells (Fig. ).
Sequence analysis of the det1::mTn3 mutant showed that transposon insertion occurred between chromosomal coordinates 557748 and 557749 on chromosome IV, allowing for the production of a truncated protein corresponding to the first 104 amino acids of Det1. Preliminary results indicated that this truncated protein acts in a dominant-negative way to inhibit sterol uptake (not shown), potentially accounting for the severity of the defect (~20-fold) in the rate of sterol accumulation in C209 cells (Fig. ) compared with the milder defect (~3-fold reduction) in the rate of biosynthetic sterol transport in det1Δ cells (Fig. ).
How might Det1 play a role in nonvesicular sterol transport? Det1 is an ~39-kDa soluble protein with a predicted abundance of ~3,000 molecules per cell (20
). GFP-tagged Det1 is localized primarily to the cytoplasm (12
), consistent with a role in sterol transport across the cytoplasm. Sequence analysis shows that Det1 has significant similarity to phosphoglycerate mutases. However, Det1 lacks a number of residues shown to be critical for catalysis (51
), and this activity (as performed by Gpm1, Gpm2, and/or Gpm3) in S. cerevisiae
has been well characterized (17
). Other than the predicted similarity to phosphoglycerate mutases, sequence analysis reveals that Det1 has a number of close homologs in other fungi (i.e., KLLA0F18810g in Kluyveromyces lactis
and ca5949 in Candida albicans
) but no clear homologs in mammals. Perhaps Det1 is involved in a yeast-specific or ergosterol-specific aspect of sterol transport or significant sequence divergence has occurred in the course of evolution of the more complex cholesterol regulatory mechanisms in higher eukaryotes. While the identification of Det1 as an important player in intracellular sterol transport opens the door to future studies on this poorly understood process, more analyses are required in order to determine the exact nature and mechanism of its involvement.