1. The Arabidopsis AtGCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides
- Author
-
Nan Yao, Jian Yin, Kai-En Li, Fang-Cheng Bi, Guang-Yi Dai, Chan Rong, Ding-Kang Chen, and Zhe Liu
- Subjects
0106 biological sciences ,0301 basic medicine ,Ceramide ,biology ,Chemistry ,Endoplasmic reticulum ,Lipid microdomain ,Cell Biology ,biology.organism_classification ,01 natural sciences ,Biochemistry ,Sphingolipid ,Glucosylceramidase ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Arabidopsis ,Arabidopsis thaliana ,Molecular Biology ,Glucosylceramides ,010606 plant biology & botany - Abstract
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 AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 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 AtGCD3 are vital for GCD activity. GFP-AtGCD3 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 gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.
- Published
- 2019