The Neurospora crassa CPS-1 polysaccharide synthase functions in cell wall biosynthesis

Abstract

The Neurospora crassa cps-1 gene encodes a polysaccharide synthase with homology to the Cryptococcus neoformans hyaluronic acid synthase Cps1p. Homologs of the cps-1 gene are found in the genomes of many fungi. Loss of CPS-1 results in a cell wall defect that affects all stages of the N. crassa life cycle, including vegetative growth, protoperithecia (female mating structure) development, and conidia (asexual spore) development. The cell wall of cps-1 deletion mutants is sensitive to cell wall perturbation reagents. Our results demonstrate that CPS-1 is required for the incorporation of cell wall proteins into the cell wall and plays a critical role in cell wall biogenesis. We found that the N. crassa cell wall is devoid of hyaluronic acid, and conclude that the polysaccharide produced by the CPS-1 is not hyaluronic acid.

Keywords: Cell wall biogenesis; Cell wall glucan; Cell wall polysaccharide; Fungal cell wall; Neurospora crassa; Polysaccharide synthase.

Fig. 1

The Δcps-1 mutant is affected in hyphal cell morphology. Pictures of the growth at the edge of wild type and Δcps-1 colonies were taken with a Cannon Power Shot A620 camera with a microscope adaptor. Both pictures were taken with at 200X magnification. The white bar represents a distance of 20 μm.

Fig. 2

RIP and complementation experiments demonstrate that loss of cps-1 is responsible for the mutant phenotype. Slants containing 1) WT, 2) Δcps-1, 3) cps-1^RIP1, and 4) Δcps-1 being complemented by a wild type copy of cps-1 are shown. The slants show 4 day-old cultures grown at room temperature.

Fig. 3

CPS-1 is a 58 kD protein. A HA-tagged version of CPS-1 was prepared as described in Materials and Methods and used to transform a Δcps-1; his-3 isolate. The presence of HA-tagged CPS-1in a cellular extract was assessed by Western blot analysis with anti-HA antibody. Lane 1) Transformant cell extract. Lane 2) wild type cell extract.

Fig. 4

Δcps-1 releases large amounts of cell wall protein into the growth medium. Wild type and Δcps-1 were grown for 24 hours in liquid Vogel's medium and harvested. Cell extracts and samples of the growth medium were subjected to SDS-PAGE and stained with Coomassie Blue. Lane M) molecular weight markers. Lane 1) 30 ug of wild type cell extract. Lane 2) Secreted wild type proteins corresponding to 30 ug of cell extract. Lane 3) 30 ug of Δcps-1 cell extract. Lane 4) Secreted proteins corresponding to 30 ug of Δcps-1 cell extract.

Fig. 5

Δcps-1 releases large amounts of ACW-1, a known cell wall protein, to the growth medium. Proteins were loaded on an SDS gel as shown in Fig. 4 and subjected to Western blot analysis for ACW-1. Lane 1) 30 ug of wild type cell extract. Lane 2) Secreted wild type proteins corresponding to 30 ug of cell extract. Lane 3) 30 ug of Δcps-1 cell extract. Lane 4) Secreted proteins corresponding to 30 ug of Δcps-1 cell extract.

Fig. 6

The Δcps-1 cell wall is deficient in cell wall proteins. The Coomassie Brilliant Blue dye binding assay was used to assess the levels of cell wall proteins in purified wild type and Δcps-1 cell walls. Increasing amounts of cell wall proteins were incubated with the dye and the amount of dye binding to cell wall proteins was determined.