In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

Abstract

Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1, Chs2 and Chs3, of which the latter accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. To fill this gap of knowledge, we performed bioinformatic analyses and protease protection assays that revealed significant information about the catalytic domain, the chitin-translocating channel and the interfacial helices in between. In particular, we identified an amphipathic, crescent-shaped α-helix attached to the inner side of the membrane that presumably controls the channel entrance and a finger helix pushing the polymer into the channel. Evidence has accumulated in the past years that chitin synthases form oligomeric complexes, which may be necessary for the formation of chitin nanofibrils. However, the functional significance for living yeast cells has remained elusive. To test Chs3 oligomerization in vivo, we used bimolecular fluorescence complementation. We detected oligomeric complexes at the bud neck, the lateral plasma membrane, and in membranes of Golgi vesicles, and analyzed their transport route using various trafficking mutants.

The localization of chitin synthase mediates the patterned deposition of chitin in developing Drosophila bristles

“…Both of these regions are thought to be cytoplasmic in yeast CHS2, although there is evidence for two 83 transmembrane domains separating the catalytic site from at least the c terminal most part of Con2 84 (Gohlke et al, 2017) . The insect cuticle CS is typically larger than most fungal CS proteins.…”

“…All CS are 88 multi-pass transmembrane proteins. The number of inferred transmembrane domains varies from ~5 to 89 18 with fungal CS proteins generally predicted to contain many fewer putative transmembrane domains 90 than insect CS proteins (Gohlke et al, 2017;Merzendorfer, 2011) (Merzendorfer and Zimoch, 2003). 91 However, since different programs predict different numbers of transmembrane domains for individual 92 CS until direct experimental data provides answers there will be uncertainty (Gohlke et al, 2017).…”

“…The number of inferred transmembrane domains varies from ~5 to 89 18 with fungal CS proteins generally predicted to contain many fewer putative transmembrane domains 90 than insect CS proteins (Gohlke et al, 2017;Merzendorfer, 2011) (Merzendorfer and Zimoch, 2003). 91 However, since different programs predict different numbers of transmembrane domains for individual 92 CS until direct experimental data provides answers there will be uncertainty (Gohlke et al, 2017). 93 Indeed, in the case of fungal chitin synthases direct experimental data established that the computer 94 programs for predicting transmembrane domains are useful but not able to accurately predict 95 membrane protein topology (Gohlke et al, 2017).…”

“…91 However, since different programs predict different numbers of transmembrane domains for individual 92 CS until direct experimental data provides answers there will be uncertainty (Gohlke et al, 2017). 93 Indeed, in the case of fungal chitin synthases direct experimental data established that the computer 94 programs for predicting transmembrane domains are useful but not able to accurately predict 95 membrane protein topology (Gohlke et al, 2017). We report here experimental evidence that the 96 amino terminus of Drosophila Kkv is in the cytoplasm and the carboxy terminus in the extracellular 97 space.…”

The deduced role of a chitinase containing two nonsynergistic catalytic domains

“…Chitin synthesis meets these criteria. The oligomerization of chitin synthase is crucial for the generation of insoluble chitin fibrils, as pre-aligned catalytic units could facilitate the proper alignment of nascent sugar chains before coalescence (Sacristan et al, 2013;Gohlke et al, 2017). Evidence of trimeric ChsB complexes from the larval midgut of Manduca sexta (tobacco hornworm) have been reported (Maue et al, 2009), which are presumed to be further oligomerized to form higher-order complexes.…”