Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis

Abstract

The internal environment of the ER is regulated to accommodate essential cellular processes, yet our understanding of this regulation remains incomplete. Cod1p/Spf1p belongs to the widely conserved, uncharacterized type V branch of P-type ATPases, a large family of ion pumps. Our previous work suggested Cod1p may function in the ER. Consistent with this hypothesis, we localized Cod1p to the ER membrane. The cod1Delta mutant disrupted cellular calcium homeostasis, causing increased transcription of calcium-regulated genes and a synergistic increase in cellular calcium when paired with disruption of the Golgi apparatus-localized Ca2+ pump Pmr1p. Deletion of COD1 also impaired ER function, causing constitutive activation of the unfolded protein response, hypersensitivity to the glycosylation inhibitor tunicamycin, and synthetic lethality with deletion of the unfolded protein response regulator HAC1. Expression of the Drosophila melanogaster homologue of Cod1p complemented the cod1Delta mutant. Finally, we demonstrated the ATPase activity of the purified protein. This study provides the first biochemical characterization of a type V P-type ATPase, implicates Cod1p in ER function and ion homeostasis, and indicates that these functions are conserved among Cod1p's metazoan homologues.

Figure 1.

Cod1p is localized to the endoplasmic reticulum. Comparison of the distribution of 3myc–Cod1p with the distribution of the ER proteins Kar2p (A) and Sec61p (B) by immunofluorescence as described in the Materials and methods. Yeast lysates prepared in the presence of EDTA (C) or magnesium (D) were fractionated on a 10–60% linear sucrose gradient. Protein from each fraction was separated by SDS-PAGE, transferred to nitrocellulose, and probed with the appropriate antibodies.

Figure 1.

Cod1p is localized to the endoplasmic reticulum. Comparison of the distribution of 3myc–Cod1p with the distribution of the ER proteins Kar2p (A) and Sec61p (B) by immunofluorescence as described in the Materials and methods. Yeast lysates prepared in the presence of EDTA (C) or magnesium (D) were fractionated on a 10–60% linear sucrose gradient. Protein from each fraction was separated by SDS-PAGE, transferred to nitrocellulose, and probed with the appropriate antibodies.

Figure 2.

Expression of calcium-responsive genes is induced in cod1 Δ . (A) Log phase cultures of wild-type (RHY1708) and cod1Δ cells (RHY1709) expressing β-galactosidase reporters were grown in YPD and assayed for β-galactosidase activity as described in the Materials and methods. (B) Log phase cultures of wild-type and cod1Δ cells were grown overnight in the presence or absence of 30 μg/ml of cyclosporin A and assayed for β-galactosidase activity. Representative experiments are shown.

Figure 3.

cod1 Δ display phenotypes specific to calcium but not to manganese. (A) Growth arrest of wild-type, cod1Δ, pmr1Δ, and cod1Δ/pmr1Δ cells (CS601A, CS601B, CS601C, and CS601D) by CaCl₂ or MnCl₂. Low-density cultures (OD₆₀₀ < .01) were grown in liquid YPD, pH 5.5, in the presence of the indicated concentrations of added CaCl₂ or MnCl₂ for 2.5 d. Growth is plotted as a percentage of the outgrowth of the untreated culture. (B) Deletion of PMR1 and COD1 causes a synergistic increase in total cellular calcium, but not manganese. Cultures were grown overnight in YPD, pH 5.5, to an optical density of approximately one and then processed as described in the Materials and methods. Representative experiments are shown.

Figure 4.

Deletion of COD1 produces phenotypes indicating the presence of misfolded proteins in the ER. (A) Growth arrest of wild-type (RHY791) and cod1-1 (RHY811) cells by tunicamycin or DTT. Low-density cultures (OD₆₀₀ < 0.01) were grown in minimal medium in the presence of the indicated concentrations of added tunicamycin or DTT. Growth is plotted as a percentage of the outgrowth of the untreated culture. (B) Wild-type (RHY1708) and cod1Δ (RHY1709) cells transformed with a plasmid expressing GFP from four unfolded protein response elements were treated for 4 h with the indicated concentrations of tunicamycin or DTT and analyzed by flow microfluorimetry. Each histogram represents 10,000 cells. (C) cod1-1 null mutants (RHY910) were mated to hac1Δ mutants (RHY1664). The diploids were sporulated and dissected on YPD plates. (D) Wild-type, hac1Δ, pmr1Δ, and hac1Δ/pmr1Δ cells (MYY290, RHY1884, RHY2179, and RHY2277) were streaked onto minimal media lacking inositol or containing 20 mM EGTA.

Figure 5.

Glycosylation and the UPR are not required for regulating Hmg2p–GFP degradation. (A) Tunicamycin does not affect the stabilization of Hmg2p–GFP by lovastatin. Early log phase cultures were grown for 4 h in 4 μg/ml tunicamycin and the presence or absence of 25 μg/ml lovastatin and subjected to flow cytometry. (B) Hmg2p–GFP regulation does not require HAC1. Early log phase cultures of wild-type (RHY872) or HAC1Δ (RHY1664) cells were grown for 4 h in the presence or absence of 25 μg/ml lovastatin and subjected to flow cytometry.

Figure 6.

Functional conservation of Cod1 between S. cerevisiae and D. melanogaster. (A) Lovastatin sensitivity of cod1Δ cells (RHY1401) transformed with a high copy plasmid expressing nothing, P _TDH3-HA::COD1, or P _TDH3-HA::dCOD1. Cultures were streaked onto plates containing 200 μg/ml lovastatin and incubated at 30°C. (B) Regulation of Hmg2p–GFP degradation in cod1-1 cells (RHY911) expressing the indicated P-type ATPases expressed from the PMA1 promoter. Early log phase cultures were grown for 4 h in the presence or absence of 25 μg/ml lovastatin and subjected to flow cytometry.

Figure 7.

The D487N mutation inactivates Cod1p. Regulation of Hmg2p–GFP in cod1Δ cells (RHY2681) transformed with a plasmid expressing P _TDH3-9HIS–HA–COD1 (COD1) or P _TDH3-9HIS–HA–COD1 with the D487N mutation (D487N). Lovastatin was added at 25 μg/ml and cultures were grown at 30°C for 4 h after the addition of lovastatin.

Figure 8.

Purification and ATPase activity of wild-type and D487N versions of 9HIS–HA–Cod1p. (A) SDS-PAGE stained with Gelcode blue. (B) Western blots with anti-HA antibodies. (C) 0.015 μg of histidine-tagged Cod1p (⋄), D487N (♦), or nothing (▵) were incubated in 50 mM Hepes/TRIS, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, and 50 μM γ³²-labeled ATP for the indicated times. (D) 0.015 μg of histidine-tagged Cod1p (⋄) was incubated for 5 min in 50 mM Hepes/TRIS, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, and the indicated amounts of γ³²-labeled ATP. Representative experiments are shown.

Figure 9.

Effect of ions on Cod1p. (A) 0.135 μg of histidine-tagged Cod1p was incubated in 1 mM EDTA, 50 mM Hepes/TRIS, 100 mM KCl, 10 mM MgCl₂, 50 μM [γ³²]ATP, and increasing amounts of CaCl₂, MnCl₂, or CuSO₄. Experiments with CuSO₄ were done without EDTA. Results are plotted with respect to the calculated free concentration of the indicated ion. (B) 0.15 μg of histidine-tagged Cod1p was incubated in 50 mM Hepes/TRIS, 100 mM KCl, 5 mM MgCl₂, and 50 μM γ³²-labeled ATP for 5 min with the addition of 100 μM of the indicated ions, or nothing. (C) 0.15 μg of Cod1p was incubated in 50 mM Hepes/TRIS, 100 mM KCl, 5 mM MgCl₂, and 50 μM γ³²-labeled ATP for 5 min in the presence of the indicated concentrations of TPEN, 1,10-phenanthroline (Phen), or EDTA. (D) 0.015 μg of Cod1p was incubated for 5 min in 50 mM Hepes/TRIS, 100 mM KCl, 1 mM EDTA, 50 μM γ³²-labeled ATP, and increasing concentrations of MgCl₂. The ATPase activity of Cod1p (⋄) is plotted with respect to the concentration of free Mg²⁺ on the primary y axis. The calculated concentration of MgATP with respect to the concentration of free Mg²⁺ is plotted as a dotted line on the secondary y axis. (E) The same data as in D, plotted on the primary y axis with respect to the calculated concentration of MgATP. The calculated concentration of free Mg²⁺ is plotted with respect to the calculated concentration of MgATP as a dotted line. Representative experiments are shown.