The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry

Abstract

Membrane asymmetry is a key organizational feature of the plasma membrane. Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that establish membrane asymmetry by translocating phospholipids, such as phosphatidylserine (PS) and phospatidylethanolamine, from the exofacial leaflet to the cytosolic leaflet. Saccharomyces cerevisiae expresses five P4-ATPases: Drs2, Neo1, Dnf1, Dnf2, and Dnf3. The inactivation of Neo1 is lethal, suggesting Neo1 mediates an essential function not exerted by the other P4-ATPases. However, the disruption of ANY1, which encodes a PQ-loop membrane protein, allows the growth of neo1Δ and reveals functional redundancy between Golgi-localized Neo1 and Drs2. Here we show Drs2 PS flippase activity is required to support neo1Δ any1Δ viability. Additionally, a Dnf1 variant with enhanced PS flipping ability can replace Drs2 and Neo1 function in any1Δ cells. any1Δ also suppresses drs2Δ growth defects but not the loss of membrane asymmetry. Any1 overexpression perturbs the growth of cells but does not disrupt membrane asymmetry. Any1 coimmunoprecipitates with Neo1, an association prevented by the Any1-inactivating mutation D84G. These results indicate a critical role for PS flippase activity in Golgi membranes to sustain viability and suggests Any1 regulates Golgi membrane remodeling through protein-protein interactions rather than a previously proposed scramblase activity.

Keywords: Golgi apparatus; phosphatidylcholine; phosphatidylethanolamine; phosphatidylserine; trafficking; transport.

Fig. 1.

PS translocation in Golgi membranes is required for viability. A: Interacting network of proteins important for membrane organization whose function in membrane asymmetry and cell viability is explored in this study. B: Neo1, Drs2, and Dnf1[N550S] can support the growth of neo1Δ drs2Δ any1Δ cells. neo1Δ drs2Δ any1Δ + pRS416-NEO1 (MTY10-615M2DS) expressing single-copy (cen) NEO1 (pRS313-NEO1), empty vector (pRS313), cen DRS2 (pRS313-DRS2), cen Drs2[QQ→GA] (pRS313-Drs2[QQ→GA]), cen DNF1 (pRS313-DNF1), or cen Dnf1[N550S] (pRS313-Dnf1[N550S]), respectively, were spotted onto minimal media plates, and the pRS416-NEO1 plasmid was counter-selected on 5-FOA. C: Loss of Kes1 failed to suppress the synthetic lethality of neo1Δ drs2Δ any1Δ. neo1Δ drs2Δ any1Δ kes1Δ + pRS416-NEO1 (MTY10-615M2DS) expressing single-copy (cen) NEO1 (pRS313-NEO1), empty vector (pRS313), cen DRS2 (pRS313-DRS2), and cen Drs2[QQ→GA] (pRS313-Drs2[QQ→GA), respectively, were spotted onto minimal media plates, and the pRS416-NEO1 plasmid was counter-selected on 5-FOA. These images are representative of four independent growth assays.

Fig. 2.

None of the Golgi P4-ATPases can individually restore membrane asymmetry when expressed in neo1Δ any1Δ drs2Δ cells. The strains indicated were grown with increasing concentrations of PapA (A) or Duramycin (B) to assess the exposure of PS or PE, respectively. Growth (OD₆₀₀) after 20 h at 30°C is plotted relative to untreated WT cells (n ≥ 4; error bars indicate ± SEM).

Fig. 3.

any1Δ genetic interactions with P4-ATPase mutants. A: any1Δ suppresses the cold-sensitive growth of drs2Δ, high-temperature growth defects of neo1-1 and neo1-2, and the synthetic lethality between drs2 and neo1^ts alleles. Both neo1-1^ts and neo1-2^ts can support the viability of any1Δ drs2Δ cells at all temperatures tested except for 37°C. B: Loss of Any1 failed to suppress the synthetic lethality of dnf1,2,3Δ drs2Δ. C: Loss of Any1 can suppress the synthetic lethality of neo1Δ lem3Δ or neo1^ts lem3Δ at all temperatures tested except for 37°C. These images are representative of four independent growth assays.

Fig. 4.

Deletion of Any1 failed to suppress the loss of PS and PE asymmetry in drs2Δ or dnf1,2Δ mutants, respectively. The strains indicated were grown with increasing concentrations of Papa A (A, C) or Duramycin (B, D) to assess the exposure of PS or PE, respectively. Growth (OD₆₀₀) after 20 h at 30°C is plotted relative to untreated WT cells (n ≥ 4; error bars indicate ± SEM).

Fig. 5.

ANY1 overexpression partially inhibits the growth of drs2Δ and dnf1,2Δ mutants but is most detrimental to neo1^ts, dop1^ts, and mon2Δ mutants. A: ANY1 overexpression completely inhibits the growth of neo1^ts any1Δ, dop1^ts any1Δ, and mon2Δ any1Δ mutants. Growth assays were performed with WT, neo1^ts any1Δ, dop1^ts any1Δ, and mon2Δ any1Δ mutants expressing empty vector or pBY011-ANY1 (P_GAL1-ANY1) at 27, 34, and 37°C. B: ANY1 overexpression partially inhibits the growth of drs2Δ and dnf1,2Δ mutants to a greater extent than WT cells. WT, drs2Δ, and dnf1,2Δ expressing empty vector or pBY011-ANY1 (P_GAL1-ANY1) were spotted onto the minimal media plates containing glucose or galactose at 30°C. These images are representative of four independent growth assays.

Fig. 6.

ANY1 overexpression induces a modest loss of PE asymmetry but does not increase PS exposure in WT or drs2Δ cells. A, B: ANY1 overexpression does not induce a loss of PS asymmetry in WT and drs2Δ cells. C, D: ANY1 overexpression induces a partial but significant loss of PE asymmetry in WT and drs2Δ cells. WT and drs2Δ cells expressing empty vector (pRS416) or pRS416-P_GAL1-ANY1 were grown to mid-log phase in the minimal media containing glucose. Cells (0.2 OD₆₀₀) were shifted to minimal medium containing galactose or glucose (control) in the presence of pore-forming toxins PapA or Duramycin. Growth relative to the vehicle control was plotted. Student’s t-test was performed for each concentration tested between WT and drs2Δ cells (n ≥ 2).

Fig. 7.

Effect of Any1 point mutations on its ability to antagonize Neo1 and inhibit the growth of P4-ATPase mutants. A: Any1[D84G] suppressed neo1Δ lethality, whereas Any1[G80R] inhibited growth comparably to WT ANY1. Plasmid shuffling assays were performed with neo1Δ any1Δ + pRS416-NEO1 (MTY10) expressing single copy NEO1 (pRS313-NEO1), empty vector (pRS313), ANY1 (pRS313-ANY1), ANY1[D84G] (pRS313-Any1[D84G]) or ANY1[G80R] (pRS313-Any1[G80R]); each strain was then spotted onto minimal media plates (SD) and the pRS416-NEO1 plasmid was counter selected on 5-fluorotic acid (5-FOA). B: Overexpression of ANY1 using pRS416-P_GAL1-ANY1 construct inhibits the growth of WT, drs2Δ, and dnf1,2Δ mutants. WT, drs2Δ, and dnf1,2Δ expressing pRS416 (empty vector) or pRS416-P_GAL1-ANY1 were spotted onto the minimal media plates containing glucose or galactose at 30°C. C, D: Growth inhibition due to ANY1 overexpression is suppressed by the D84G mutation but not the G80R mutation.

Fig. 8.

Any1 physically interacts with Neo1, and the [D84G] mutation abolishes this interaction. A: Expression of Neo1-FLAG, Any1-GST, and Any1[D84G]GST in cells used for coimmunoprecipitation experiments. neo1Δ any1Δ (MTY10S) cells expressing pRS315-NEO1 or pRS315-NEO1-5XFLAG along with pRS313-ANY1-GST or pRS313-Any1[D84G]GST were lysed in SDS/urea and probed for Neo1-FLAG and Any1-GST expression. B: Any1 interacts with Neo1. neo1Δ any1Δ (MTY10S) cells expressing pRS315-NEO1 or pRS315-NEO1-5XFLAG along with pRS313-ANY1-GST or pRS313-Any1[D84G]GST were spheroplasted and lysed in the presence of detergent. Immunoprecipitations were performed using these total lysates in the presence or absence of the DSP cross-linker. Immunoprecipitations were also probed for Arf1 as a negative control. Immunoprecipitations were performed two independent times.