To further understand the mechanism of the void zone formation, we created a series of deletion mutants in the background of the cho1Δ or glucose-repressible PGAL1-3HA-CHO1 mutations, and examined their effect on the generation of the void zone (Fig. 7A). Various genes involved in sterol trafficking were tested, and the results indicate that kes1Δ (osh4Δ) mildly and arv1Δ significantly reduced the void zone formation. Kes1 is one of the yeast oxysterol-binding proteins that exchanges sterols for PI(4)P between the lipid membranes (Jiang et al., 1994; de Saint-Jean et al., 2011). Arv1 was implicated in the GPI-anchor biosynthesis and transport and in intracellular sterol distribution (Kajiwara et al., 2008; Beh and Rine, 2004). Both Kes1 and Arv1 are involved in the sterol transport; however, their contribution to the sterol transport to the PM is very low (Georgiev et al., 2011, 2013). These proteins regulate sterol organization in the PM; the mutations influence the sensitivity to the sterol-binding drugs and sterol-extraction efficiency of methyl-β-cyclodextrin (MβCD) (Georgiev et al., 2011, 2013). These differences in sterol organization may influence the formation of the void zone. Npc2 is an orthologue of Niemann–Pick type C protein and plays an essential role, together with Ncr1, in sterol insertion into the vacuolar membrane from the inside of the vacuole, which is required for the formation of the raft-like vacuolar domain during lipophagy in the stationary phase (Tsuji et al., 2017). However, deletion of Npc2 did not influence generation of the void zone or formation of the V–V contacts associated with the protein-depleted vacuolar domain (Fig. S5). This result suggests that the void zone and vacuolar domains at the V–V contact are formed independently of Ncr1- and Npc2-mediated sterol transport.

The void zone formation was strongly suppressed by deletion of vacuolar proteins, Vma2 and Fab1. Vma2 is a subunit of the V-ATPase that regulates pH homeostasis (Marshansky et al., 2014). Consistent with this observation, other genes involved in pH homeostasis (NHA1, NHX1, RIM21 and RIM101) were required for the void zone formation (Fig. 7A, pH regulation) (Sychrová et al., 1999; Brett et al., 2005; Obara et al., 2012). Thus, we examined the effect of pH of the medium on the formation of the void zone. The formation of the void zone was strongly suppressed by increasing pH in the medium from 6.6 to 7.5 (Fig. 7B). On the other hand, the low pH medium (pH 4.0) slightly increased the formation of the void zone. Interestingly, the proportion of cells with V–V contacts was significantly reduced in the low pH medium (Fig. 7C). The mechanism of these pH-dependent phenomena is unclear although they may be important in assessment of the molecular basis of the formation of the void zones and V–V contacts.

Fab1 is a phosphatidylinositol 3-phosphate [PI(3)P] 5-kinase that generates phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] (Cooke et al., 1998). PI(3,5)P2 functions as a signal lipid in intracellular homeostasis, adaptation and retrograde membrane trafficking (Jin et al., 2016). We speculated that defects in retrograde transport may indirectly influence the void zone formation in the PM, and thus examined various genes involved in membrane transport. Strikingly, conserved protein complexes, retromer, CORVET, HOPS and ESCRT, were required for the void zone formation (Fig. 7A,D). As the name implies, the Vps proteins belonging to these complexes were identified from mutants defective in vacuolar protein sorting (VPS) (Robinson et al., 1988; Rothman et al., 1989). Dysfunction of these complexes perturbs intracellular vesicle trafficking (Schmidt and Teis, 2012; Balderhaar and Ungermann, 2013; Burd and Cullen, 2014), which may influence the PM recycling of cargo and lipids involved in the void zone formation. Similarly, proteins involved in the retrograde transport, such as the SNAREs Pep12 and Tlg2, the epsin-like adaptors Ent3 and Ent5, and the clathrin adaptors Gga1 and Gga2, were required for the void zone formation. The dynamin-like GTPase Vps1, Arf-like GTPase Arl1 and Rab6 GTPase homologue Ypt6 are known to be closely related to the membrane trafficking (Vater et al., 1992; Li and Warner, 1996; Rosenwald et al., 2002). Consistent with this notion, vps1Δ, arl1Δ, and ypt6Δ inhibited the void zone formation. Impaired membrane transport in the inner membrane system can be manifested as changes in the PM lipid organization and/or defects in the pH control. However, Apl2 and Apl1, subunits of the adaptor complexes AP-1 and AP-2, respectively, had little contribution to the void zone formation, presumably because these mutants had insignificant disruption of the membrane trafficking compared to the effects of ent3Δ, ent5Δ and gga1Δ gga2Δ (Yeung et al., 1999; Sakane et al., 2006; Morvan et al., 2015). Deletion of APL5, which encodes the subunit of AP-3 responsible for the transport from the Golgi to the vacuole (Dell'Angelica, 2009), slightly reduced void zone formation, suggesting the importance of the vacuolar functions for void zone formation.

Deletion of the autophagy-related genes (atg1Δ, atg10Δ, atg12Δ and atg15Δ) did not influence void zone formation. ATG1 is one of the core ATG genes (Mizushima et al., 2011). The void zone was generated and the V–V contact with the vacuolar microdomain was observed in the absence of Atg1 (Fig. 7A; Fig. S5). This result suggests that void zone formation is independent of autophagy, consistent with our notion that direct or indirect effects on lipid organization in the PM (e.g. via the Vps pathway) are influencing the void zone formation.

We also examined the effect of mutations in the flippase-related proteins. The deficiency of Cdc50 or Any1 and Cfs1, localized in endosomes and the TGN, had little effect on the void zone formation (Saito et al., 2004; van Leeuwen et al., 2016; Yamamoto et al., 2017); however, disruption of Lem3 localized in the PM completely suppressed the generation of the void zone (Kato et al., 2002). The flippase complexes between Lem3 and Dnf1 or Dnf2 translocate glycerophospholipids to the cytoplasmic leaflet of the lipid bilayer (Pomorski et al., 2003; Saito et al., 2004; Furuta et al., 2007). We assumed that disruption of the phospholipid asymmetry by lem3Δ may influence ergosterol behaviour in the PM. To test this hypothesis, we examined the sensitivity to the antifungal ergosterol-binding drug amphotericin B (AmB) (Kamiński, 2014). The results indicate that lem3Δ is highly sensitive to AmB, which is detected in the cho1Δ background cells, and this effect was reversed by addition of erg6Δ, which causes defects in ergosterol biosynthesis (Fig. 7E). Thus, the disruption of phospholipid asymmetry alters the ergosterol distribution in the PM, thereby suppressing void zone formation (see Discussion).

Although it is not yet clear through which mechanisms some of these additive mutations inhibit the void zone formation, these findings may help to elucidate the nature of the void zone in the future.