Oligomerization of the chitin synthase Chs3 is monitored at the Golgi and affects its endocytic recycling

Abstract

SummaryChs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP-1-dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N-terminal cytosolic region. However, the truncated Δ126Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI-and Rer1-dependent manner. A subset of the oligomerization-deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N-terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly.

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

“…Previous studies performed in insect and fungal systems provided in vitro evidence that chitin synthases form di- or oligomeric complexes [29,30,31,32]. Based on these studies, we examined Chs3 oligomerization in vivo using BiFC analysis [36].…”

“…Previous Y2H and crosslinking experiments with truncated Chs3 versions narrowed down the self-interaction site to an N-terminal region of Chs3 between amino acid positions 1–126 [31]. An additional self-interaction site was proposed based on the results from Y2H studies for the region between amino acid positions 226 and 452 [30].…”

“…Previous dithiobis(succinimidyl propionate)-cross-linking studies had suggested that Chs3 di-/oligomerization occurs already in the membranes of the Golgi and the ER [31]. Therefore, we addressed the question whether we would be able to track Chs3 di-/oligomerization in vivo along its trafficking route from the ER to the cell surface in a wildtype genetic background as well as in several deletion mutants that are defective in Chs3 trafficking.…”

“…A yeast-two-hybrid (Y2H) study [29] reported homo-dimerization of Chs3 involving a dimerization domain that comprises the first 700 amino acids. In another Y2H study, the dimerization domain was narrowed down to the regions between amino acids 226 and 452 [30] and the N-terminal region comprising the first 171 amino acids [31]. Additional evidence that chitin synthases form oligomeric complexes were derived from biochemical studies in insects, that reported successful purification of active, oligomeric complexes of the midgut-specific chitin synthase 2 from Manduca sexta larvae (MsChs2) [32].…”

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.…”