Role of four calcium transport proteins, encoded by nca-1, nca-2, nca-3, and cax, in maintaining intracellular calcium levels in Neurospora crassa

Abstract

We have examined the distribution of calcium in Neurospora crassa and investigated the role of four predicted calcium transport proteins. The results of cell fractionation experiments showed 4% of cellular calcium in mitochondria, approximately 11% in a dense vacuolar fraction, 40% in an insoluble form that copurifies with microsomes, and 40% in a high-speed supernatant, presumably from large vacuoles that had broken. Strains lacking NCA-1, a SERCA-type Ca(2+)-ATPase, or NCA-3, a PMC-type Ca(2+)-ATPase, had no obvious defects in growth or distribution of calcium. A strain lacking NCA-2, which is also a PMC-type Ca(2+)-ATPase, grew slowly in normal medium and was unable to grow in high concentrations of calcium tolerated by the wild type. Furthermore, when grown in normal concentrations of calcium (0.68 mM), this strain accumulated 4- to 10-fold more calcium than other strains, elevated in all cell fractions. The data suggest that NCA-2 functions in the plasma membrane to pump calcium out of the cell. In this way, it resembles the PMC-type enzymes of animal cells, not the Pmc1p enzyme in Saccharomyces cerevisiae that resides in the vacuole. Strains lacking the cax gene, which encodes a Ca(2+)/H(+) exchange protein in vacuolar membranes, accumulate very little calcium in the dense vacuolar fraction but have normal levels of calcium in other fractions. The cax knockout strain has no other observable phenotypes. These data suggest that "the vacuole" is heterogeneous and that the dense vacuolar fraction contains an organelle that is dependent upon the CAX transporter for accumulation of calcium, while other components of the vacuolar system have multiple calcium transporters.

Fig. 1.

The growth of N. crassa is inhibited by high concentrations of calcium. Cells were grown for 3 days at 25°C in Vogeloid medium (pH 5.8 or 7.5) as described in Materials and Methods.

Fig. 2.

The accumulation of calcium in mycelia is dependent on the concentration of calcium in the medium. Wild-type strain 74A was grown for 25 h at 30°C in Vogel's medium N as described in Materials and Methods. (A) Uptake is expressed as nmol per 6 ml culture. (B) Uptake is expressed as percentage of total calcium in the medium.

Fig. 3.

Distribution of calcium in cell fractions from wild-type strain 74A. The cell fractionation procedure is described in Materials and Methods. Note that because of small measurement errors in some steps of the procedure, the numbers do not total exactly 100%. The microsomal fraction contains membranes from broken vacuoles, the ER, and the Golgi apparatus. The 100K supernatant contains soluble material from broken organelles and the cytosol.

Fig. 4.

Accumulation of calcium in wild-type strain 74A and in strains lacking calcium transporters. Mycelia were grown in Vogel's medium N for 24 h at 30°C as described in Materials and Methods.

Fig. 5.

Effect of calcium on the growth of strains lacking calcium transporters. Growth was measured after 2 days at 30°C in liquid Vogel's medium N, pH 7.5, as described in Materials and Methods. Panel A shows results for wild-type 74A and single mutant strains; panel B shows results for strains with multiple mutations.

Fig. 6.

Effect of calcium on the growth of strains lacking both cax and nca-2 genes. Growth was measured after 2 days in liquid Vogel's medium N, pH 7.5, as described in Materials and Methods. Note that the x axis has a logarithmic scale.

Fig. 7.

Morphology of hyphal growth of wild-type strain 74A and of strains lacking transporters. Strains were grown on plates with Vogel's medium N and 2% agar for 24 h (or 48 h for the Δvma-3 strain) at 30°C. The morphologies of hyphal growth were similar in all strains except for the Δvma-3 strain, which had a highly compact and highly branched pattern of growth. In the panel on the right, wild-type 74A and the Δnca-2 strain were grown in tubes with 2 ml of Vogel's medium N at 30°C for 3 days.