Cation diffusion facilitator Cis4 is implicated in Golgi membrane trafficking via regulating zinc homeostasis in fission yeast

Abstract

We screened for mutations that confer sensitivities to the calcineurin inhibitor FK506 and to a high concentration of MgCl(2) and isolated the cis4-1 mutant, an allele of the gene encoding a cation diffusion facilitator (CDF) protein that is structurally related to zinc transporters. Consistently, the addition of extracellular Zn(2+) suppressed the phenotypes of the cis4 mutant cells. The cis4 mutants and the mutant cells of another CDF-encoding gene SPBC16E9.14c (we named zrg17(+)) shared common and nonadditive zinc-suppressible phenotypes, and Cis4 and Zrg17 physically interacted. Cis4 localized at the cis-Golgi, suggesting that Cis4 is responsible for Zn(2+) uptake to the cis-Golgi. The cis4 mutant cells showed phenotypes such as weak cell wall and decreased acid phosphatase secretion that are thought to be resulting from impaired membrane trafficking. In addition, the cis4 deletion cells showed synthetic growth defects with all the four membrane-trafficking mutants tested, namely ypt3-i5, ryh1-i6, gdi1-i11, and apm1-1. Interestingly, the addition of extracellular Zn(2+) significantly suppressed the phenotypes of the ypt3-i5 and apm1-1 mutant cells. These results suggest that Cis4 forms a heteromeric functional complex with Zrg17 and that Cis4 is implicated in Golgi membrane trafficking through the regulation of zinc homeostasis in fission yeast.

Figure 1.

Mutation in the cis4⁺ gene causes MgCl₂- and immunosuppressant-sensitive phenotypes. Wild-type (wt), cis4-1, or Δcis4 cells transformed with the multicopy vector pDB248 or the vector containing the cis4⁺ (SPAC17D4.03c) gene were streaked onto the plates as indicated and then incubated for 4 d at 27°C.

Figure 2.

Cis4 is homologous to zinc transporters and the phenotypes of cis4 mutants are zinc suppressible. (A) Comparison of the amino acid sequences among fission yeast Cis4, budding yeast Msc2p, and human ZnT7. Amino acid sequences are aligned using the Clustal W program. Filled boxes indicate the mutated amino acids. (B) Phenotypes of the cis4 mutants are zinc suppressible. A 5-μl sample of cells was spotted onto the plates as indicated in serial 10-fold dilutions starting with OD₆₆₀ = 0.3 of log-phase cells and then incubated for 4 d at 27°C. (C) Simultaneous expression of budding yeast MSC2 and ZRG17 genes suppressed the MgCl₂-sensitive phenotype of Δcis4 cells. A 5-μl sample of Δcis4 cells expressing various genes were spotted onto EMM plates as indicated and incubated for 4 d at 27°C.

Figure 3.

The cis4 and zrg17 mutant cells share common and nonadditive zinc-suppressible phenotypes (A), and Cis4 and Zrg17 physically interact (B). (A) Wild-type, Δcis4, Δzrg17, or Δcis4Δzrg17 cells were spotted onto the plates as indicated and then incubated for 4 d at 27°C. (B) In cells expressing GFP-Cis4 from the chromosomally integrated gene, GST, or GST-Zrg17 was expressed from the harboring plasmid at 27°C. GST-tagged protein was pulled down by glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies.

Figure 4.

Cis4 protein localizes at the cis-Golgi. Cells expressing Cis4-RFP were transformed with the vector expressing Rer1-GFP (A) or Krp1-GFP (B). Cells were grown to early log phase in EMM containing 4 μM thiamine. Bar, 10 μm. DIC, differential interference contrast (microscopy).

Figure 5.

The cis4 mutants show defects in cell wall integrity, secretion, and membrane trafficking. (A) MgCl₂-sensitive phenotype of cis4 mutants were osmoremedial. Wild-type, cis4-1, cis4-2, or Δcis4 cells were spotted onto the plates as indicated and then incubated for 4 d at 27°C. (B) The addition of Zn²⁺ suppressed the increased septation index of Δcis4 cells. Wild-type (wt) or Δcis4 cells were incubated in EMM with or without 2 mM ZnSO₄ at 27°C. Septation index (■, percentage of cells with a septum) of log phase cells was monitored. At least 50 cells were observed at one time in each experiment. The data represent mean ± SD of 10 determinations. The reduction in the septation index by the addition of Zn²⁺ was statistically significant (p < 0.005). (C) The cis4 mutants are hypersensitive to micafungin. The cis4 mutants showed hypersensitivity to micafungin, a (1,3)-β-d-glucan synthase inhibitor. Cells were spotted onto YPD plates and YPD plus 0.9 μg/ml micafungin and incubated at 27°C for 4 d. (D) MgCl₂-sensitive phenotype of cis4 mutants were suppressed by the expression of constitutively active calcineurin. Wild-type cells or cis4-1 mutant cells transformed with a control vector, the vector containing the full-length ppb1⁺ gene (CN full), or the vector containing the constitutively active truncated calcineurin gene (CNΔC) were spotted onto each YPD plate as indicated and incubated for 4 d at 27°C. (E) The cis4 deletion markedly impaired the secretion of acid phosphatase. Wild-type or Δcis4 cells were assayed for secreted (left panel) and accumulated (right panel) acid phosphatase activities. The data represent mean ± SD of five determinations. (F) Defective localization of GFP-Syb1 in Δcis4 cells. Wild-type (wt) and Δcis4 cells expressing GFP-Syb1 were cultured in EMM medium containing 4 μg/ml thiamine at 27°C and were examined by fluorescence microscopy. Top panel, arrows indicate GFP fluorescence associated with the plasma membrane. Bar, 10 μm. Bottom panel, cells with clear fluorescence at the cell membrane were counted. At least 50 cells were observed at one time in each experiment. The data represent mean ± SD of five determinations.

Figure 6.

Genetic interaction between cis4 and membrane-trafficking mutants. (A) The ypt3-i5Δcis4, ryh1-i6Δcis4, gdi1-i11Δcis4, or apm1-1Δcis4 double mutants showed more marked temperature sensitivity than the single mutants. Wild-type (wt), Δcis4, ypt3-i5, ypt3-i5Δcis4, ryh1-i6, ryh1-i6Δcis4, gdi1-i11, gdi1-i11Δcis4, apm1-1, and apm1-1Δcis4 cells were spotted onto YPD plates and then incubated for 4 d at the temperatures as indicated. (B) The cis4 deletion markedly enhanced the sensitivity of the ypt3-i5 cells to Zymolyase. Wild-type, Δcis4, ypt3-i5 or ypt3-i5Δcis4 cells exponentially growing in YPD medium were harvested and incubated with β-glucanase (Zymolyase) at 27°C with vigorous shaking. Cell lysis was monitored by the measurement of optical density at 660 nm (the value before adding the enzyme was taken as 100%). The data represent mean ± SD of five determinations.

Figure 7.

Effect of the addition of extracellular Zn²⁺ on the membrane-trafficking mutants. (A) Effect of Zn²⁺ on temperature sensitivity of the membrane-trafficking mutants. Wild-type, ypt3-i5, ryh1-i6, gdi1-i11, or apm1-1 mutant cells were spotted onto the plates and then incubated for 3 or 4 d at the temperatures as indicated. (B) Effect of Zn²⁺ on FK506 sensitivity of the membrane-trafficking mutants. Wild-type, ypt3-i5, ryh1-i6, gdi1-i11, or apm1-1 mutant cells were spotted onto the plates as indicated and then incubated for 4 d at 27°C. (C) Effect of Zn²⁺ on high septation index of the membrane-trafficking mutants. Septation index of the membrane-trafficking mutants cells were monitored as described in Figure 5B. The data represent mean ± SD of 10 determinations. The reduction in the septation index by the addition of Zn²⁺ was statistically significant (p < 0.005) in the case of ypt3-i5 and apm1-1 mutant cells. (D) Effect of the expression of constitutively active calcineurin on the temperature-sensitive phenotypes of the membrane-trafficking mutants. Membrane-trafficking mutant cells transformed with a control vector or with the vector containing the constitutively active truncated calcineurin gene (CNΔC) were spotted onto each YPD plate and incubated as indicated for 3–4 d.

Figure 8.

Cartoon about relationship between zinc and calcineurin in the regulation of membrane trafficking. Schematic diagram is based on the collective findings in this report combined with what has been reported in the literature. Block arrow with a question mark indicates unknown pathways and the larger arrow indicates stronger effects.