Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha

Abstract

Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole.

Fig 1. Growth of the pmr1-Δ and ret1-27 mutants on SD* medium supplemented with different concentrations of MnCl₂.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. The experiment was performed using serial dilutions of cell suspensions (S3 Fig) and a representative dilution is shown in this figure. pmr1-Δ, a subclone of the 1MA27/12/GP1 strain lacking the PMR1 containing plasmid; the PMR1, 1MA27/12/GP1 strain, ret1-27, the 64MA70QAL strain; RET1, the 64MA70QA-RET strain.

Fig 2. Rescue of growth of the pmr1-Δ ret1-27 double mutant by CaCl₂ and MnCl₂.

Since the ret1-27 pmr1-Δ double mutant was inviable on regular media, it was obtained from the MC39-MOX strain, which carried a PMR1-contaning plasmid. To allow the MC39-MOX and MC39-RET-MOX strains to lose the PMR1-contaning plasmid, they were streaked on YPD plate supplemented with 10 mM CaCl₂. Equal amounts of cells from single colonies obtained on this medium were suspended in sterile water and spotted onto test plates. Growth of only one subclone for each case is shown in the figure. Growth of three additional subclones is shown in S4 Fig pmr1-Δ ret1-27, a MC39-MOX subclone lacking the plasmid; PMR1 ret1-27, a MC39-MOX subclone retaining the plasmid; pmr1-Δ RET1, a MC39-RET-MOX subclone lacking the plasmid; PMR1 RET1, a MC39-RET-MOX subclone retaining the plasmid.

Fig 3. Rescue of growth of the pmr1-Δ pmc1-Δ double mutant by CaCl₂ and MnCl₂.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. The experiment was performed using serial dilutions of cell suspensions (S5 Fig) and a representative dilution is shown in this figure. PMR1 pmc1-Δ and pmr1-Δ pmc1-Δ, the 1MA77/12/GAP2-Δpmc strain with or without the PMR1-containing plasmid, respectively; PMR1 PMC1 and pmr1-Δ PMC1, the 1MA77/12/GAP2 strain with or without the PMR1-containing plasmid, respectively.

Fig 4. Effect of the vps35-Δ mutation on growth of strains with or without the PMR1 gene.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. The experiment was performed serial dilutions of cell suspensions (S6 Fig) and a representative dilution is shown in this figure. pmr1-Δ vps35-Δ, the 1MA27/12/GP1-Δvps35 strain lacking the plasmid with PMR1; vps35-Δ, the 1MA27/12/GP1-Δvps35 strain, pmr1-Δ VPS35, the 1MA27/12/GP1 strain lacking the plasmid with PMR1; PMR1 VPS35, the 1MA27/12/GP1 strain.

Fig 5. Sensitivity of the ret1-27 pmc1-Δ double mutant to a shortage or excess of Ca²⁺ in culture medium.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. Ca²⁺ shortage was achieved by addition of EGTA to SD* medium. Excess of Ca²⁺ was achieved by supplementing YPD with CaCl₂. The experiment was repeated with serially diluted cell suspensions (S7 Fig). ret1-27 pmc1-Δ, the 64MA70QA-Δpmc strain; pmc1-Δ, the 64MA70Q-RET-Δpmc strain; ret1-27, the 64MA70QAL strain; WT, the 64MA70QL-RET strain.

Fig 6. Effect of inactivation of PMC1 on the level of Pmr1 in the ret1-27 mutant strain and in the strain bearing the RET1 wild-type allele.

Proteins from cell lysates were resolved by SDS PAGE and transferred to nitrocellulose membrane, which was then divided in two parts at the level of the 80 kDa marker band. The upper part was stained with antiserum against H. polymorpha Pmr1, while the lower part was stained with antibody against tubulin used as a loading control. ret1-27 pmc1-Δ, the 64MA70QA-Δpmc strain; pmc1-Δ, the 64MA70Q-RET-Δpmc strain; ret1-27, the 64MA70QAL strain; WT and WT 1/2, undiluted and two-fold diluted sample of the 64MA70QL-RET strain, respectively.

Fig 7. Immunoblot analysis of CPY from culture supernatants (A) and cell lysates (B).

ret1-27 vps35-Δ, the 64MA70UA-Δvps35 strain; ret1-27 vps10-Δ, the 64MA70UA-Δvps10 strain; vps35-Δ, the 64MA70U-RET-Δvps35 strain; vps10-Δ, the 64MA70U-RET-Δvps10 strain; ret1-27, the 64MA70UAL strain; WT, the 64MA70UA-RET strain. +EndoH, samples treated with endoglycosidase H. X3, an overexposed image (~3-fold longer time) of the "vps35-Δ" lane. 1/8, an underexposed (~8-fold shorter time) image of the “ret1-27” and “WT” lanes.

Fig 8. Immunoblot analysis of Gas1 from cell lysates.

ret1-27 vps35-Δ, the 64MA70UA-Δvps35 strain; ret1-27 vps10-Δ, the 64MA70UA-Δvps10 strain; vps35-Δ, the 64MA70U-RET-Δvps35 strain; vps10-Δ, the 64MA70U-RET-Δvps10 strain; ret1-27, the 64MA70UAL strain; WT, the 64MA70UA-RET strain. +EndoH, samples treated with endoglycosidase H.

Fig 9. Immunoblot analysis of uPA from culture supernatants.

uPA, the strains 64MA70UAL (ret1-27), 64MA70UA-RET (WT), 64MA70U-RET-Δvps10 (vps10-Δ), and 64MA70U-RET-Δvps35 (vps35-Δ), expressing the wild-type uPA; uPA-Q³⁰², the strains 64MA70QAL (ret1-27), 64MA70QA-RET (WT), and 64MA70Q-RET-Δvps10 (vps10-Δ), expressing the unglycosylated uPA-Q³⁰² mutant protein. Samples with the wild-type uPA were treated with EndoH prior to electrophoresis.

Fig 10. Cell Tracker Blue staining of vacuoles.

ret1-27 vps35-Δ, the 64MA70UA-Δvps35 strain; ret1-27 vps10-Δ, the 64MA70UA-Δvps10 strain; vps35-Δ, the 64MA70U-RET-Δvps35 strain; vps10-Δ, the 64MA70U-RET-Δvps10 strain; ret1-27, the 64MA70UAL strain; WT, the 64MA70UA-RET strain. The white bar corresponds to 5 μm. Arrows indicate the additional compartments stained like the vacuole.