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doi: 10.1242/10.1242/jcs.00307

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Journal of Cell Science 116, 1095-1105 (2003)
doi: 10.1242/jcs.00307

Research Article

Spatial and cellular localization of calcium-dependent protease (CDP II) in Allomyces arbuscula

Mukti Ojha* and Francisco Barja

Laboratoire de Bioénergétique et Microbiologie, Université de Genève, 3 Place de l'Université, CH-1211 Genève 4, Switzerland

* Author for correspondence (e-mail: Mukti.Ojha{at}bota.unige.ch)

Accepted 5 December 2002

Immunogold labeling of calcium-dependent neutral protease II (CDPII) with specific antibodies in near median longitudinal ultrathin sections of Allomyces arbuscula showed that the enzyme is predominantly localized in the growing hyphal and rhizoidal apices. The tips in both cell type had more enzyme than the distal regions and showed a gradient distribution. Labeling of the ultrathin sections and western blot analysis of purified subcellular fractions showed that CDPII is mainly cytosolic. Catalytic activity of the enzyme measured with synthetic substrate (Bz-Arg-pNA) showed that 90% of its activity is present in the soluble fraction, although a small amount is associated with the nuclei (0.2%), plasma membranes (0.7%) and microsomes (3.9%). This association is discussed in the context of the functional role of the enzyme and its possible localized activation. Western blot analysis of the crude extract and indirect immunofluorescence of the fixed permeabilized hypahe after treatment with CDPII showed that the {alpha}-tubulin is a specific target of the enzyme.

Key words: Allomyces arbuscula, Calcium-dependent neutral protease, Immunogold labeling, Intracellular localization, Hyphal tips, {alpha}-tubulin a specific target






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