The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo.
Little is known about the assembly and turnover of cellulose synthase complexes commonly called rosettes. Recent work indicates that rosette assembly could involve the dimerization of CesA (cellulose synthase catalytic subunit) proteins regulated by the redox state of the CesA zinc-binding domain (ZnBD). Several studies in the 1980s led to the suggestion that synthase complexes may have very short half-lives in vivo, but no recent work has directly addressed this issue. In the present work, we show that the half-life of cotton fiber GhCesA1 protein is <30 min in vivo, far less than the average membrane protein. We also show that the reduced monomer of GhCesA1 ZnBD is rapidly degraded when exposed to cotton fiber extracts, whereas the oxidized dimer is resistant to degradation. Low rates of degradation activity were detected in vitro by using extracts from fibers harvested during primary cell-wall formation, but activity increased markedly during transition to secondary cell-wall synthesis. In vitro degradation of reduced GhCesA1 ZnBD is inhibited by proteosome inhibitor MG132 and also by E64 and EGTA, suggesting that proteolysis is initiated by cysteine protease activity rather than the proteosome. We used a yeast two-hybrid system to identify a putative cotton fiber metallothionein and to confirm it as a protein that could interact with the GhCesA1 ZnBD. A model is proposed wherein active cellulose synthase complexes contain CesA proteins in dimerized form, and turnover and degradation of the complexes are mediated through reductive zinc insertion by metallothionein and subsequent proteolysis involving a cysteine protease.
Jacob-Wilk
Debora
D
Section of Plant Biology, One Shields Avenue, University of California, Davis, CA 95616, USA.
Kurek
Isaac
I
Hogan
Patrick
P
Delmer
Deborah P
DP
D001671:Biochemistry / Q000379:methods; D002473:Cell Wall / Q000378:metabolism; D019281:Dimerization; D004533:Egtazic Acid / Q000737:chemistry / Q000494:pharmacology; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D003368:Gossypium / Q000201:enzymology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers
D011994:Recombinant Proteins; D004533:Egtazic Acid; D005964:Glucosyltransferases; C478648:cellulose synthase; D010100:Oxygen