ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification

Abstract

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

Keywords: ABC transporter; cholesteryl ester; lipid droplet; sterol transport.

Figure 1.

Exogenous cholesterol esterification is independent of the endosomal and vacuolar pathways. A) Exogenous cholesterol redistribution. Cells of the indicated genotype were grown anaerobically in the presence of 4 µg/ml NBD cholesterol for 3 d or stained with FM4-64 (100 µg/ml at 30°C for 90 min), and assessed by fluorescence or light (DIC, differential interfering contrast) microscopy. NBD cholesterol-derived fluorescence is restricted to plasma membranes and subcellular lipid droplets (arrow) in control cells and to the plasma membrane of acyltransferase-deficient (are1Δ are2Δ) cells. B) Esterification of exogenous cholesterol. Cells of the indicated genotypes were grown anaerobically in the presence of 0.01 μCi/ml [4-¹⁴C]cholesterol. Esterification is presented as the mean esterified [4-¹⁴C]cholesterol (percentage of control ± se). In control cells, 78% of exogenous sterol was converted to steryl ester. Statistically significant difference from control is indicated. **P < 0.01 by unpaired Student’s t test.

Figure 2.

Multimerization of sterol esterification enzymes. A) Expression of yeast sterol esterification enzymes. Microsomal proteins from SCY059 (are1Δ are2Δ) strains expressing the indicated genes were separated by 6.5% SDS-PAGE and blotted with αAre2p or αHA antisera. B) Physical interactions of yeast sterol esterification enzymes. Microsomal proteins were immunoprecipitated with anti-HA antibody, eluted, separated by 6.5% SDS-PAGE, and blotted with αAre2p or αHA antisera. Co-IP, coimmunoprecipitation.

Figure 3.

Sterol homeostasis in mutants lacking putative Are2p-interacting partner proteins. The indicated genes were deleted in upc2-1 strains or grown anaerobically as mutations in control backgrounds to facilitate uptake of 0.01 μCi/ml [4-¹⁴C]cholesterol. The data (2 independent sets of triplicates) reflect the mean esterified [4-¹⁴C]cholesterol (percentage of control ± se). In control cells, 80% of exogenous sterol was converted to steryl ester. Statistically significant differences from control are indicated. *P < 0.05 and **P < 0.01 by unpaired Student’s t test.

Figure 4.

Are2p, Aus1p, and Pdr11p form a complex in vivo. Aus1-YFP (A and C) or Pdr11-YFP (B and D) strains were transformed with a vector control or pRS424/Are2-HA. Microsomes were solubilized and immunoprecipitated with GFP-conjugated agarose beads followed by denaturing gel electrophoresis and immunoblotting with an α-HA antisera. ns, a nonspecific cross-reacting species. The extracts prior to immunoprecipitation are shown in the input lanes. A and B) ABC transporter and acyltransferases have a shared microenvironment. C and D) ABC transporter and acyltransferases form a complex separated by <15 Å. Cell extracts were incubated with the membrane-permeable cleavable cross-linker DSP, prior to immunoprecipitation, SDS-PAGE resolution, and immunoblotting.

Figure 5.

Membrane properties of yeast ABC-sterol transporters. A) Aus1p and Pdr11p form a complex. The membrane yeast 2-hybrid (iMYTH) testing was carried out as described in Materials and Methods. The AUS1 bait strain and an unrelated control bait strain were transformed with NubG/NubI control prey plasmids or with prey plasmid expressing a NubG-tagged PDR11 fragment (corresponding to aa 326–518). Cells were spotted onto transformation selection medium (T, synthetic medium lacking leucine) or interaction selection medium (I, synthetic medium lacking tryptophan, leucine, adenine, and histidine). B) Fluorescent localization of Aus1-YFP and Pdr11-YFP in upc2-1 strains. Fluorescence (YFP) and differential interfering contrast (DIC) images are shown. Punctate fluorescence at the plasma membrane/cell periphery is indicated (arrows). C) Aus1-YFP and Pdr11-YFP localize to cold DRMs. Cold detergent-resistant proteins from strains expressing the indicated proteins were prepared as in Materials and Methods, resolved by 4–15% gradient SDS-PAGEs and immunoblotted with the indicated antisera (αPma1; DRM marker, αGFP for the indicated ABC transporter-YFP fusions).

Figure 6.

Yeast ABC transporters modulate membrane association of sterol esterification enzymes. In control cells (A), Are1p and Are2p localize to cold detergent-soluble (fractions 7 and 8) or -resistant (peak fractions 2 and 3) microdomains, respectively, coincident with the indicated marker proteins (αPma1; DRM, αSec22; non-DRM). In the absence of either ABC transporter (B), the Are2 acyltransferase becomes detergent soluble (peak fractions 7 and 8). Cold detergent-resistant proteins from indicated strains were prepared as in Materials and Methods, resolved by 4–15% gradient SDS-PAGEs, and immunoblotted with indicated antisera to Pma1p, Sec22, or HA (for Are1p and Are2p).

Figure 7.

Mammalian ABCG1 and ACAT1 localize to DRMs in murine tissues. DRMs were isolated and analyzed as described in Materials and Methods; tissue homogenates were solubilized with cold 1% Triton X-100 and fractionated by sucrose gradient centrifugation. Isolated fractions were run on 4–15% gradient SDS-PAGEs and immunoblotted with the indicated antisera. A) Acat1 and ABCG1 colocalize with flotillin-1 (a DRM marker) in control murine lung. B) ACAT activity is predominantly associated with plasma membrane (PM) DRMs in fractions isolated from murine brain. Plasma membrane DRMs were isolated and analyzed as described in Materials and Methods. Statistically significant differences between detergent-soluble and -insoluble assays are indicated. **P < 0.001.

Figure 8.

Detergent-resistant properties of ACAT1 are altered in the absence of ABCG1 and ABCG4 in murine brain tissues. A) DRMs were isolated from brain homogenates from animals of the indicated genotypes with cold 1% Triton X-100, fractionated by sucrose gradient centrifugation and resolved by SDS-PAGEs, and probed with the indicated antisera. B) ImageJ analysis of immunoblots. Flotillin-1 (a DRM marker) expression peaks in fractions 4–7 for all genotypes. In control tissues, Acat1 expression peaks in DRM fractions (4–7). However, Acat1 expression is present in detergent-resistant and detergent-soluble fractions in mG1^−/−, mG4^−/−, and mG1^−/− mG4^−/− tissues.

Figure 9.

Models for coupling of exogenous sterol transport and esterification in S. cerevisiae. Exogenous sterol is imported into the cell via the DRM-residing ABC transporters, Aus1p and Pdr11p. Upon influx, sterols are rapidly and efficiently esterified by Are2p, which also resides in a DRM. The newly synthesized steryl ester (SE) then accumulates in CLDs. A) Are2p serves as a bridge between the ER and plasma membrane (PM) at ER–plasma MCSs. In the absence of either ABC transporter, Are2p relocalizes to a detergent-soluble domain, and the fraction of exogenous sterol that is esterified decreases. By contrast, the minor O-acyltransferase, Are1p, resides solely in the ER in detergent-soluble membranes where it predominantly esterifies sterol biosynthetic intermediates as they are synthesized. B) The transporters and acyltransferases exist as multimeric complexes at the plasma membrane in DRMs. Loss of Aus1p or Pdr11p results in the altered residence of Are2p in a plasma membrane detergent-soluble fraction and a concomitant decrease in the percentage of cellular steryl esters.