The GCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides.

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified , one of four homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant GCD3 has a low for the fluorescent lipid C-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, -butyldeoxynojirimycin, inhibits GCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in GCD3 are vital for GCD activity. GFP-GCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in mutants. Our results indicate that GCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.