Affiliation:
1. Department of Molecular, Cell, and Developmental Biology, University of California; Santa Cruz, California 95064
2. Department of Biochemistry and Biophysics, Oregon State University, ALS 2011, Corvallis, Oregon 97331-7305
Abstract
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 Ca
2+
-ATPase of animal cells, colocalized 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 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 Ca
2+
/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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
Cited by
95 articles.
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