Proteolytic processing of fungal and insect proteins involved in chitin synthesis

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Title: Proteolytic processing of fungal and insect proteins involved in chitin synthesis
Authors: Meissner, Derek Gilbert
Thesis advisor: apl. Prof. Dr. Hans Merzendorfer
Thesis referee: Prof. Dr. Jürgen J. Heinisch
Abstract: In this thesis the regulation of chitin synthases by proteolytic activation has been analyzed in yeast and insects. It was shown that the solubilized chitin synthase 2 of Manduca sexta (MsChs2) is an oligomeric complex of about 10 nm in diameter. In contrast to MsChs2 in membrane fractions, it can be activated by trypsin and chymotrypsin in the solubilized and purified state. In yeast, proteolytic activation of chitin synthases has been described almost 40 years ago. However, no protease has been identified stimulating chitin synthesis in vivo. Recently, Martinez-Rucobo et al. (2009) demonstrated, that the chitin synthase 2 (Chs2) of Saccharomyces cerevisiae is activated by a still unknown soluble endogenous protease. A global screening for protein-protein interactions indicated that the metalloprotease Ste24 interacts with chitin synthase 3 (Chs3). Ste24 is a membrane-integral CaaX protease residing in the endoplasmic reticulum (ER). In yeast, the only known substrate of Ste24 is the mating factor a (MFa) precursor. The interaction between Ste24 and Chs3 was verified by yeast two hybrid analysis and the interacting domains were mapped. Further investigations focused on the characterization of Ste24’s influence on chitin synthesis. Growth tests demonstrated that ste24D mutants are resistant to Calcofluor White (CFW). Mutant cells expressing a catalytically inactive version of Ste24 were also CFW resistant and showed a decrease in chitin levels. Overexpression of STE24 resulted in hypersensitivity to CFW and a slight increase in chitin levels. The CFW phenotype of ste24D cells could be rescued by its human and insect orthologues. Additionally, Chs3-GFP localized less frequently at the bud neck in ste24D cells. Although Chs3 binds to Ste24, it appears not to be a substrate of this protease. Instead Ste24 modulates the chitin synthesis by cleaving the CaaX motif of prenylated Chs4, a known activator of Chs3, since in cells expressing non-prenylated Chs4, deletion or overexpression of Ste24 had no influence on chitin synthesis. Moreover, the data suggests that Chs3 and Ste24 form a complex in the ER that facilitates proteolytic activation of Chs4, a known activator of Chs3 with a C-terminal CaaX motif, leading to a more efficient localization of Chs3 at the plasma membrane.
Subject Keywords: Yeast; Chitin; Protease; ste24; chs3; Manduca; Chs2; bud neck
Issue Date: 18-Nov-2010
License name: Namensnennung-Keine Bearbeitung 3.0 Unported
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Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB05 - E-Dissertationen

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