A PhotoClick cholesterol‐based quantitative proteomics screen for cytoplasmic sterol‐binding proteins in Saccharomyces cerevisiae

Abstract

Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins.Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs.We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking. K E Y W O R D Sclick chemistry, membrane, non-vesicular transport, proteomics, sterol

TOR complex 2 is a master regulator of plasma membrane homeostasis

“…Given our findings [ 211 ] that Ypk1-mediated phosphorylation of Lam2 and Lam4 inhibits their function in retrograde sterol transport, we sought to understand the mechanism of this negative regulation. We found [ 222 ], in agreement with a prior study [ 58 ] that, at ER-plasma membrane contact sites, Lam2 and Lam4 associate with two paralogous β-propeller (WD40 repeat) proteins Laf1/Ymr102c and Dgr2/Ykl121w, one of which was detected as a sterol-binding protein [ 223 ]. Using fluorescent tags, we showed that Lam2 and Lam4 remain at ER-plasma membrane contact sites when Laf1 and Dgr2 are absent, whereas neither Laf1 nor Dgr2 remain at ER-plasma membrane contact sites when Lam2 and Lam4 are absent [ 222 ].…”

TORC2-Dependent Ypk1-Mediated Phosphorylation of Lam2/Ltc4 Disrupts Its Association with the β-Propeller Protein Laf1 at Endoplasmic Reticulum-Plasma Membrane Contact Sites in the Yeast Saccharomyces cerevisiae

“…Moreover, given the genetic and other evidence we have presented here that Laf1 and Dgr2 have a function in retrograde sterol transport, in conjunction with Lam2 and Lam4, we suspect that the residues lining the central cavity in the Laf1 and Dgr2 ten-bladed β-propellers selectively accommodate ergosterol. Fully consistent with this supposition, using a photoactivatable sterol derivative, Laf1 (Ymr102c) was detected recently as a sterol-binding protein in yeast extracts [68]. The fact that Laf1 is at least three times more abundant than Dgr2 possibly explains why Dgr2 was not detected by the same approach.…”

Synthesis of a Side Chain Alkyne Analogue of Sitosterol as a Chemical Probe for Imaging in Plant Cells

“…1 H NMR (400 MHz, CDCl 3 ) δ 5.29-5.19 (m, 1H, H-22), 5.08 (dd, J = 15.1, 9.2 Hz, 0.46H, H-23), 4.95 (dd, J = 15.1, 9.4 Hz, 0.61H, H-23), 4.17-4.03 (m, 2H, H-1 ′ ), 3.32 (d, J = 0.7 Hz, 3H, OCH 3 ), 2.77 (t, J = 2.4 Hz, 1H, H-6), 2.40-2.28 (m, 1H, H-2 ′ ), 2.14-1.57 (m, 8H), 1.53-0.96 (m, 24H), 0.90-0.79 (m, 6H, H-1, H-3, H-9 and H-29), 0.72 (d, J = 2.6 Hz, 3H, H-18), 0.65 (t, J = 4.4 Hz, 1H, H-4), 0.43 (dd, J = 8.0, 5.0 Hz, 1H, H-4). 13 Ethyl (24R,25RS)-6β-Methoxy-3α,5-cyclo-5α-stigmastan-26-oate (12) This compound was synthesized from ∆22 analogue 11 (0.45 g, 0.93 mmol) via catalytic hydrogenation using Pd on a charcoal catalyst, according to a procedure from the literature [26] as follows: yield: 0.40 g, 0.82 mmol, 88%. 1 (24R,25RS)-6β-Methoxy-3α,5-cyclo-5α-stigmastan-26-ol (13) To a stirred suspension of LiAlH 4 (0.30 g, 8.0 mmol) in dry THF (30 mL) under nitrogen atmosphere, a solution of ethyl ester 12 (0.39 g, 0.80 mmol) in dry THF (20 mL) was added, and the mixture was stirred over night at room temperature.…”

“…In the final step, both the 3-OH and the Δ5 m ties were regenerated from the steroid i-methyl ether in an 86% yield via the treatm with p-toluenesulfonic acid in dioxane. The obtained target compound FB-DJ-1 (4) wa expected, about a 1:1 mixture of epimers at C-25; neither this compound nor the pre sors (12,13,15) were separable via chromatography on a preparative scale (Scheme 1…”

“…Click chemistry has meanwhile found ample applications in steroid chemistry, with applications in organic synthesis, material science, drug discovery, and bioconjugation chemistry [9,11]. Recently, CuAAC has also been combined with photo-crosslinking ("PhotoClick cholesterol") for the identification of sterol-binding proteins in yeast cells by means of quantitative proteomics [12].…”