The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa

Abstract

Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neumspora crassa. A single band of approximately 11 0 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.

A Class-V Myosin Required for Mating, Hyphal Growth, and Pathogenicity in the Dimorphic Plant PathogenUstilago maydis [W]

“…The ability to grow in a polar manner also was reflected by the polar organization of F-actin patches at apical growth sites ( Figure 2C; anti-actin antibodies) and by caps of newly deposited chitin at these tips ( Figure 2D; wheat germ agglutinin [WGA] staining). This distribution of chitin coincided with the polar localization of chitin synthases that was detected with a broadly cross-reactive anti-Chs2p antibody (Sietsma et al, 1996; kindly provided by H. Sietsma [University of Groningen, Haren, The Netherlands] and H. Deising [MartinLuther-University, Halle, Germany]) ( Figure 2E). …”

“…At the permissive temperature (20 Њ C), myo5 ts mutants grew slightly slower ( Figure 2A) and were thicker, but they did grow by polar budding and (E) Localization of chitin synthase detected with a cross-reactive antibody raised against Chs2p from S. cerevisiae (Sietsma et al, 1996) showed no cytokinesis defect ( Figure 2F1; for comparison, see control in Figure 2B1). After a shift to restrictive conditions (34ЊC), cells enlarged further and exhibited the first defects in cell separation, with fine wall fibers connecting the almost separated cells (Figure 2F2, arrow; 1.5 h at 34ЊC).…”

“…Monoclonal anti-actin antibody (1:100, clone JLA20; Oncogene, Darmstadt, Germany), anti-␣-tubulin (1:200; Oncogene), and chitin synthase polyclonal antibody (1:200; Sietsma et al, 1996) were diluted in PBS and 0.1% milk powder. Secondary antibodies (Jackson, West Grove, PA) were used at 1:200.…”

Polar Localizing Class V Myosin Chitin Synthases Are Essential during Early Plant Infection in the Plant Pathogenic FungusUstilago maydis

“…Therefore, it is expected that CHSs are concentrated at the growing apex, but direct evidence for such localization in fungal hyphal tips is rare (Archer, 1977;Sietsma et al, 1996). We fused GFP to the endogenous copy of all CHSs and show here that the fusion proteins of the class IV enzymes Chs5 and Chs7, as well as the class V members Chs6 and Mcs1, are concentrated at the growing tip of yeast-like cells and hyphae, whereas Chs3 and Chs4, as well as Chs5-, Chs6-, Chs7-, and Mcs1-GFP/YFP fusion proteins localized to septa, where new cell wall is synthesized.…”

“…The synthesis of chitin is mediated by membrane-bound chitin synthases (CHSs) that locate to specialized transport vesicles, the chitosomes (Bracker et al, 1976;Leal-Morales et al, 1994;Sietsma et al, 1996) or to the plasma membrane (Duran et al, 1979;Leal-Morales et al, 1988;Martinez and Gozalbo, 1994). The yeast Saccharomyces cerevisiae contains three CHSs that have cell cycle-specific functions in septum formation and during budding (Cabib et al, 2001).…”