| Summary: | 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.
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