Comparison of the vacuolar membrane ATPase of Neurospora crassa with the mitochondrial and plasma membrane ATPases.

Abstract Plasma membrane Mg2+, Ca2+ ATPases were isolated from Dunaliella parva by differential centrifugation and subsequent sucrose gradient centrifugation and analyzed for their properties with special emphasis on ecophysiological requirements of this extremely salt-tolerant alga. Most properties (Vmax- and KM-values, substrate specificity, vanadate and DES sensitivity, resistance against ouabain) indicate that the ATPases of the plasma membrane of D. parva are basically of the same type as that found in the plasma membrane of other algae or higher plants. However, some interesting deviations from the normal characteristics of plasma membrane ATPases of plants were observed for the Dunaliella ATPases. These modifications partially may reflect adaptations of the ATPase and/or the microenvironment of the ATPase to the highly saline environment of this alga; 1) The plasma membrane ATPase of D. parva requires unusually high concentrations of divalent cations (up to 100 mM Mg2+ or Ca2+) for maximal activity. Both cations can substitute for each other. 2) The plasma membrane ATPase of D. parva is extremely resistant against salt. It was stimulated by NaCl or KC1 at concentrations up to 800 mM , whereas at higher salt concentrations the enzyme was inhibited. However, about 2.5 M NaCl was required for halfmaximal inhibition of ATPase activity. 3) The ATPase was inhibited by inhibitors of anion transport such as SITS and D ID S . which suggests direct or indirect involvement of ATPase in anion transport. The possible functions of the plasma membrane ATPases are discussed with special emphasis on problems related to the hypersaline environment of this alga.

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Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00174-09 ◽

2009 ◽

Vol 8 (12) ◽

pp. 1845-1855 ◽

Cited By ~ 73

Author(s):

Barry J. Bowman ◽

Marija Draskovic ◽

Michael Freitag ◽

Emma Jean Bowman

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Neurospora Crassa ◽

Fluorescent Protein ◽

Protein A ◽

Vacuolar Membrane ◽

Red Fluorescent Protein ◽

Animal Cells ◽

Marker Proteins ◽

Hyphal Tip

ABSTRACT We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca2+-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca2+/H+ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 μm from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

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