Chitin Translocation Is Functionally Coupled with Synthesis in Chitin Synthase

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Chitin is a crucial extracellular polysaccharide synthesized by membrane-embedded chitin synthases (CHS).
• The mechanism by which synthesized chitin crosses the membrane to extracellular locations is not fully understood.

Purpose of the Study:

• To investigate the mechanism of chitin translocation across the membrane.
• To determine if chitin synthase (CHS) itself is responsible for chitin translocation.
• To characterize the function and structure of Phytophthora sojae chitin synthase (PsCHS).

Main Methods:

• Purification and reconstitution of Phytophthora sojae chitin synthase (PsCHS) into proteoliposomes (PLs).
• Assessing chitin synthesis and translocation within proteoliposomes.
• Attempting structural resolution of PsCHS in chitin-bound and UDP/Mn2+-bound states.

Main Results:

• PsCHS functions as a processive glycosyltransferase, rapidly producing and binding highly polymerized chitin.
• PsCHS was demonstrated to be a bifunctional enzyme, both synthesizing and translocating chitin.
• Reconstituted PsCHS translocated newly synthesized chitin into the lumen of proteoliposomes, protecting it from degradation.
• A high-resolution structure of the UDP/Mn2+-bound state provided insights into the enzyme's transmembrane channel.

Conclusions:

• Phytophthora sojae chitin synthase (PsCHS) is bifunctional, responsible for both chitin synthesis and translocation.
• Chitin translocation by PsCHS is tightly coupled to its synthesis process.
• PsCHS is indispensable for moving chitin across the membrane to extracellular locations.