EC Accepts, published online ahead of print on 2 October 2009

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Eukaryotic Cell doi:10.1128/EC.00174-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa

Barry J. Bowman^*, Marija Draskovic, Michael Freitag, and Emma Jean Bowman

Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA 95064; and Department of Biochemistry and Biophysics, Oregon State, University, ALS 2011, Corvallis, OR 97331-7305

^* To whom correspondence should be addressed. Email: bowman{at}biology.ucsc.edu.

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 Neurospora 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 Ca^2+-ATPase of animal cells, co-localized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca^2+-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They co-localized 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 Ca⁺⁺/H⁺ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. Approximately 50-100 μm from the tip we observed 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.