Sterol biosensor reveals LAM-family Ltc1-dependent sterol flow to endosomes upon Arp2/3 inhibition

Abstract

Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell.

Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b

“…This is consistent with the inefficient GRAMD1-dependent transport of cholesterol observed with these mutations in vitro (Fig 4C). Yeast mutants that lack GRAMD1 homologs (Lam/Ltc proteins) are highly susceptible to treatment with the polyene antibiotic, Amphotericin B, compared to WT cells (Gatta et al, 2015;Marek et al, 2020). Amphotericin B binds PM sterols and eventually causes cell death by various mechanisms, including sequestration of PM sterols and the formation of non-selective ion pores (Kinsky, 1970;Wang et al, 2018).…”

“…These membrane contact sites play critical roles in many aspects of cell physiology, including the non-vesicular exchange/delivery of specific lipids (e.g., cholesterol) by lipid transfer proteins (Elbaz & Schuldiner, 2011;Lev, 2012;Drin, 2014;Holthuis & Menon, 2014;Gatta et al, 2015;Murley et al, 2015;Saheki et al, 2016;Saheki & De Camilli, 2017a,b;Antonny et al, 2018;Luo et al, 2018;Jeyasimman & Saheki, 2019;Nishimura & Stefan, 2019;Petrungaro & Kornmann, 2019;Wong et al, 2019;Lees & Reinisch, 2020;Meng et al, 2020;Prinz et al, 2020). We and others recently found that a family of evolutionarily conserved and ER-anchored sterol transfer proteins, the GRAMD1s/Asters (GRAMD1a/Aster-A, GRAMD1b/Aster-B, and GRAMD1c/Aster-C) (the Lam/Ltc proteins in yeast), mediate nonvesicular sterol transport from the PM to the ER at ER-PM contact sites, thereby contributing to sterol homeostasis (Gatta et al, 2015;Sandhu et al, 2018;Naito et al, 2019;Marek et al, 2020). GRAMD1s possess an N-terminal GRAM domain, an evolutionarily conserved PH-like domain (Begley et al, 2003;Tong et al, 2018), a cholesterolharboring StART-like domain, and a C-terminal transmembrane domain that anchors the protein to the ER.…”

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

“…A D4 derivative, D4H (D4 D434S ), has been developed as a more sensitive probe; it binds to liposomes containing 20-30% cholesterol mole concentration [48, 77]. Although D4H has recently been used to detect the PM sterol in fission yeast [78], D4H has not been applied to budding yeast. We generated two fluorescent protein-conjugated D4Hs, GFP-D4H and GFPenvy-D4H, in which GFPenvy is a photostable dimeric GFP derivative [79, 80].…”

Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex

“…Lam2p localizes to spots both in cytoplasmic ER and where cER and PM are associated, though distinct from where other functional tethers are observed (Gatta et al, 2015;Quon et al, 2018). In fission yeast, the Lam homolog Ltc1p is also localized to sites on perinuclear ER and the cell cortex, where it is involved in retrograde sterol transfer (Marek et al, 2020). At the cell cortex, Ltc1p facilitates an actin-dependent sterol transfer either directly from the PM to endosomes, or indirectly through the ER as an intermediate (Marek et al, 2020).…”

“…In fission yeast, the Lam homolog Ltc1p is also localized to sites on perinuclear ER and the cell cortex, where it is involved in retrograde sterol transfer (Marek et al, 2020). At the cell cortex, Ltc1p facilitates an actin-dependent sterol transfer either directly from the PM to endosomes, or indirectly through the ER as an intermediate (Marek et al, 2020).…”