Table 1.
Neurospora crassa strains used in this study.
Figure 1.
Phenotypic characterization of N. crassa mss-4.
The growth of N. crassa wild type, the mss-4(18-2) mutant [45], and a complemented strain mss-4(18-2)compl ectopically expressing wild type MSS-4 on solid media was monitored by macroscopic examination after 24 h at 37°C (A). The increase in colony diameters of growing cultures over time was determined (B). Errors (SD) are too small to be seen at the scale given. The hyphal morphologies of wild type, the mss-4(18-2) mutant or mss-4(18-2)compl grown at room temperature or 2h and 8 h after shifting to 37°C, were microscopically characterized, as indicated (C). Germination of wild type (D) and mss-4(18-2) conidia was documented after 18 h at 37°C (E). Note the highly contorted and abnormal morphology of the mss-4(18-2)-strain. A strain carrying a deletion of the entire MSS-4 locus (Δmss-4) failed to establish hyphae upon germination from ascospores (black) and, in contrast to wild type (F) was not viable (G, H). Experiments were repeated independently five times with similar results.
Figure 2.
Biochemical characterization of recombinant MSS-4(86-1012) in vitro.
MSS-4(86-1012) was heterologously expressed in E. coli. Recombinant extracts (10 µg of total protein) were incubated with 5 µg of lipid substrates in the presence of γ[32P]ATP. Phosphorylated lipids were extracted and separated by TLC. Images represent phosphoimager signals of radiolabeled lipids. Migration of lipid products was interpreted according to comigration with unlabeled authentic standards as previously described [71]. Phosphorylated lipid products were formed in vitro by MSS-4(86-1012) from different phosphatidylinositolmonophosphate-substrates, as indicated (A). Recombinant maltose-binding protein (MBP) served as a negative control; recombinant human PIP-kinase IIβ [56] served as a positive control for conversion of PtdIns5P. The phosphoimages presented in (A) were quantified (B). Specific activities were calculated using total protein content in the recombinant extracts. Data represent means ± SD of three independent experiments. The preference of MSS-4(86-1012) for PtdIns3P or PtdIns4P was tested in a competition experiment presenting lipids as individual substrates or as an equimolar mixture, as indicated (C). Phosphoimages are representative for at least three independent experiments.
Figure 3.
The N. crassa mss-4 mutant has reduced PI4P 5-kinase activity at 37°C.
The capability of membranes isolated from N. crassa wild type, mss-4(18-2) or mss-4(18-2)compl grown at 16°C or at 37°C to phoshorylate PtdIns4P was determined. Membrane preparations containing 10 µg of total protein were incubated with 5 µg of lipid substrates in the presence of γ[32P]ATP. Phosphorylated lipids were extracted and separated by TLC. Images represent phosphoimager signals of radiolabeled lipids. Migration of lipid products was interpreted according to comigration with unlabeled authentic standards as previously described [71]. Formation of PtdIns(4,5)P2 in the indicated N. crassa strains (A). The image is representative for three independent experiments. Phosphoimages presented in (A) were quantified (B). Specific activities were calculated using total protein content in the recombinant extracts. Data represent means ± SD from three independent experiments.
Figure 4.
mss-4 encodes an enzyme carrying a substitution of a conserved tyrosine residue for asparagine that results in reduced catalytic activity.
The genomic loci of two N. crassa mss-4 mutant strains were sequenced and the same nucleotide exchange 2248 T to A was found, resulting in an amino acid substitution from tyrosine 750 to asparagine. The domain structure of the deduced MSS-4 protein is presented in overview (A). The exchange Y750N is located in the C-terminal portion of the catalytic domain (arrowhead). NTD, N-terminal domains; Dim, Dimerization domain; Cat, catalytic domain; CTD, C-terminal domains. An alignment of partial amino acid sequences of various phosphatidylinositolmonophosphate kinases indicates that tyrosine 750 of NcMSS4 is strictly conserved in all sequences analyzed, including representatives from human, Drosophila melanogaster, A. thaliana and S. cerevisiae (B). According to the 3D-structure of the human PIP-kinase IIβ [56], the position of tyrosine 403 of the human enzyme, which corresponds to Y750 in MSS-4 from N. crassa, is in the substrate binding pocket (C). PtdIns, substrate analogon; ATP, position of ATP. Structure detail according to pdb entry 1bo1 [56]. Formation of PtdIns(4,5)P2 by MSS-4(86-1012) or MSS-4(86-1012;Y750N) was tested at 16°C or at 37°C, as indicated (D). Recombinant AtPIP5K2 carrying the corresponding exchange Y738N was tested in vitro for PtdIns(4,5)P2 for comparison. Wild type AtPIP5K2 was used as a positive control, as indicated. All activity tests were carried out using 10 µg of total bacterial protein and 5 µg of lipid substrate. (E) Expression of recombinant variants of MSS-4(86-1012) or AtPIP5K2 compared in (D) were tested by separating E. coli protein extracts by SDS-PAGE. Gels were stained with coomassie (left panels) or subjected to immunodetection using an anti-MBP antiserum (α-MBP; right panels), as indicated. Closed arrowheads indicate migration of MSS-4(86-1012), MSS-4(86-1012) Y750N, At-PIP5K2 or AtPIP5K2 Y738 N, as indicated. Open arrowheads indicate the migration of a 116 kDa size-marker. All experiments were performed three times with similar results.
Figure 5.
Distribution of a high-affinity fluorescent probe for PtdIns(4,5)P2 in N. crassa hyphae.
The fluorescence distribution of PLCδ1-PH-EYFP was imaged in transgenic N. crassa hyphae. The reporter decorated the plasma membrane at different developmental stages, including germinating conidia (A), and accumulated at sites of hyphal branching or emergence (B). Reporter fluorescence was also present in the plasma membrane of hyphal tips (C). The reporter associated strongly with constricting septa (D). Decoration of septa (open arrowheads) and emerging hyphal tips (closed arrowhead) could be observed in the same cell (D). Overall plasma membrane association of the reporter, strong association with septa and cytosolic puncta were observed also in older, larger hyphae (E). Bars, 5 µm (A-C; F); 20 µm (E).
Figure 6.
N. crassa MSS-4 localizes as a subapical membrane-associated ring and to constricting septa.
MSS-4 fusion proteins carrying N- or C-terminal fluorescence-tags were expressed in N. crassa hyphae or in tobacco pollen tubes, and the fluorescence distribution was monitored by confocal microscopy. GFP-MSS-4 associated with a ring-like subapical plasma membrane domain in N. crassa hyphae (A). Plasma membrane association of GFP-MSS-4 was patchy (inset). Arrowheads indicate patches. Subapical membrane localization was reduced upon expression of MSS-4(86-1012)-EYFP (B) and displayed enhanced association with cytosolic filaments or endomembrane-structures (inset). Arrowheads indicate possible filaments. MSS-4-EYFP associated with a ring-like subapical plasma membrane domain in tobacco pollen tubes (C). AtPIP5K5:EYFP localized in a ring-like subapical plasma membrane domain in tobacco pollen tubes (D) and is shown for comparison. GFP-MSS-4 also decorated constricting septa (E). Left panels, GFP; mid panels, Calcofluor; right panels, merged images. Images are representative for >100 transgenic cells observed for each expression. Bars, 10 µm.
Figure 7.
Localization of GFP-MSS-4 in tips of communicating cells during chemotropic growth and cell fusion.
Cells expressing GFP-MSS-4 were imaged in a time-lapse experiment spanning 30 min. Selected time points are shown, as indicated. Left panels, GFP; right panels, bright field. Note the increased accumulation of GFP-MSS-4 at the contact point and during generation of the fusion pore. Images are representative for 20 fusion events observed. Bars, 5 µm.