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doi: 10.1242/10.1242/jcs.00307

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

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

View larger version (129K): [in a new window]   Fig. 1. Nomarski image of actively growing Allomyces arbuscula in liquid medium (YPSs) showing two oppositely growing poles. (A) General view of a young thallus representing two polarized cell types, hyphae (h) and rhizoids (r). (B) Rhizoidal pole. (C,D) Hyphal poles showing an apical exclusion zone containing a Spitzenkörper (s) and a sub-apical region containing nuclei (n) and mitochondria (m). Bar, 10 µm.

View larger version (127K): [in a new window]   Fig. 2. Immunogold-labeled CDP II in a near median longitudinal section of hyphal apex of Allomyces arbuscula seen in an electron micrograph. (A) General view showing an apico-basal gradient of the enzyme. 1-5 correspond to the zones used for quantification of labels (see Table 2). (B) Higher magnification of the proximal tip region corresponding to zone 1. (C) Higher magnification of the distal region corresponding to zone 3 rich in mitochondria. (D) Higher magnification of zone 4 with nuclei. m, mitochondria; v, vacuole; n, nucleus.

View larger version (168K): [in a new window]   Fig. 5. Immunogold-labeled CDP II in thin sections of nuclei, mitochondria and membranes seen in an electron micrograph. (A,B) Labeling of in situ and purified mitochondria, respectively. (C,D) Labeling of in situ and purified nuclei, respectively. (E) Higher magnification of a portion of D. (F,G) Labeling along the plasma membrane (pm) in a section of hyphal tip and rhizoidal tip, respectively.

View larger version (116K): [in a new window]   Fig. 3. Immunogoldlabeled CDP II in a ultrathin longitudinal section of rhizoidal apex of Allomyces arbuscula seen in an electron micrograph. (A) General view showing abundance of the enzyme in the tip region. (B) Higher magnification of the 0-1.84 µm tip region. Plasma membrane (pm) (note the accumulation of the enzyme along the plasma membrane). m, mitochondria; mt, microtubule.

View larger version (83K): [in a new window]   Fig. 4. Control experiments for immunogold labeling of CDP II in the ultrathin sections of hyphal apices of Allomyces arbuscula. (A,B) The incubation mixture was stripped-off for CDP-II-specific antibodies by pre-incubation with purified antigen at antibody-antigen ratio of 1:0.8 and 1:1 ratio respectively. (C) Pre-immune serum was used as primary antibody.

View larger version (58K): [in a new window]   Fig. 6. Western blot analysis of CDP II in different subcellular fractions. Cell-free extract (300 g supernatant, 180 µg), nuclear (154 µg), mitochondrial (145 µg), plasma membrane (165 µg) and soluble (185 µg) protein fractions were electrophoresed in 11% SDS-PAGE and immunoblotted as described in Materials and Methods. Lanes: (1) cell-free extract; (2) nuclear; (3) mitochondrial; (4) plasma membrane; (5) soluble protein fraction.

View larger version (66K): [in a new window]   Fig. 7. Proteolysis of tubulins in the crude extract of A. arbuscula revealed by immunoblotting. 75 µg crude extract proteins were mixed with 3 µg CDP II, made up to a total volume of 120 µl with reaction buffer containing 5 mM Ca2+ and 10 mM ß-mercaptoethanol and incubated at 37°C. 20 µl samples were withdrawn at intervals, added with 5 µl sample buffer and processed for western blotting as described in Materials and Methods. (A,B) Ponceau-stained proteins on nitrocellulose membrane after SDS-PAGE and electro-transfer. The lanes a-g correspond to the time course reaction with the CDP II as shown below (C,D). (C) Crude soluble protein digest immunoblotted with -tubulin antibodies. (a) 0 minute incubation; (b) 10 minute incubation; (c) 20 minute incubation; (d) 30 minute incubation; (e) 60 minute incubation; (f) soluble proteins without enzyme; (g) soluble proteins + enzyme minus calcium. (D) Same as above but revealed with ß-tubulin antibodies

View larger version (81K): [in a new window]   Fig. 8. Distribution and in situ digestion of tubulins in the growing hyphae of A. arbuscula. Samples were prepared as described in Materials and Methods. (A) Distribution of -tubulin; (B) digestion of -tubulin by CDP II; (C) distribution of ß-tubulin; (D) digestion of ß-tubulin by CDP II.

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