Chitin Synthesis in Yeast: A Matter of Trafficking

Abstract

Chitin synthesis has attracted scientific interest for decades as an essential part of fungal biology and for its potential as a target for antifungal therapies. While this interest remains, three decades ago, pioneering molecular studies on chitin synthesis regulation identified the major chitin synthase in yeast, Chs3, as an authentic paradigm in the field of the intracellular trafficking of integral membrane proteins. Over the years, researchers have shown how the intracellular trafficking of Chs3 recapitulates all the steps in the intracellular trafficking of integral membrane proteins, from their synthesis in the endoplasmic reticulum to their degradation in the vacuole. This trafficking includes specific mechanisms for sorting in the trans-Golgi network, regulated endocytosis, and endosomal recycling at different levels. This review summarizes the work carried out on chitin synthesis regulation, mostly focusing on Chs3 as a molecular model to study the mechanisms involved in the control of the intracellular trafficking of proteins.

Keywords: chitin synthase; intracellular traffic; yeasts.

Figure 1

Structure of the chitin synthase (Chs) proteins. (A) Classification of the different Chs proteins based on their amino acid sequences and predicted secondary structures. Note that these proteins are expected to be type II integral transmembrane proteins. The different domains of each protein are indicated together with the conserved catalytic regions (gray dots). Posttranslational modifications are indicated based on the high-throughput analysis performed for the Saccharomyces cerevisiae proteome. Phosphorylation (P), ubiquitination (U), and N-glycosylation (N) are noted. (B) Detailed structure of the S. cerevisiae Chs3 protein with its three cytosolic domains and a luminal region that is N-glycosylated. The transmembrane domains are depicted with their predicted architecture, including the proposed site for palmitoylation (Pal). All the phosphorylation (S/T) and ubiquitination (K) sites that were experimentally verified are indicated. The lower part of the figure shows all the protein regions that were shown to be required for interaction with the indicated complexes or proteins. Note that most of them are located at the N-terminal cytosolic region of the protein, which serves as a major platform mediating protein interactions. (C)The predicted AlphaFold model structure for Chs3. This model confirms the proposed structure with a luminal region (upper part) and three cytosolic regions with some domains highly exposed and thus susceptible to performing protein–protein interactions. The four TM domains that cross the membrane are neatly visible and are labeled as in (B). See the text for more detailed descriptions of the roles of each domain.

Figure 2

A visual scheme for chitin synthase 3 (Chs3) trafficking. Chs3 is synthesized in the endoplasmic reticulum (ER) where it is folded and later transported to Golgi in COPII vesicles. Misfolded proteins can be detected in the Golgi and returned to the ER via COPI vesicles. Chs3 is sorted in the trans-Golgi network (TGN) using exomer in order to be transported to the plasma membrane (PM) in a polarized way. On this journey, it interacts with Chs4, which elicits chitin synthase activity, allowing its anchorage to the neck region through specific machinery. Subsequently, the protein is endocyted, being recycled to the TGN from the early endosomal compartment by the AP-1 complex. In the absence of this recycling, Chs3 can escape to the PM though an alternative route. Only a small amount of the protein, which was ubiquitinated at the PM, is recognized by the ESCRT complex and delivered to the vacuole for degradation. Part of this protein can be recycled back to the TGN by retromer, avoiding degradation. Because of these efficient recycling mechanisms, most Chs3 accumulates in the TGN, forming a dedicated intracellular reservoir known as chitosomes. The early endosomal (EE) compartment has been depicted here for clarity, but its existence in yeast is questionable, and it is very likely that recycling by the AP-1 occurs within the TGN boundaries. The width of the arrows reflects the intensity of the traffic. The images show Chs3- GFP localization in the wild type (WT) and the indicated mutants: Chs3 accumulates normally at the neck region and in intracellular vesicles (chitosomes), while images from the mutants show the accumulation of the protein at the different intracellular compartments. chs7∆: ER; chs5∆: TGN/chitosomes; end∆: PM; vps27∆: endosome E; vps35∆: vacuole.