Characterization of a CLE processing activity.
Proteins containing a conserved motif known as the CLE domain are found widely distributed across land plants. While the functions of most CLE proteins are unknown, specific CLE proteins have been shown to control shoot meristem, root and vascular development. This has been best studied for CLV3 which is required for stem cell differentiation at shoot and flower meristems. In vivo evidence indicates that the CLE domain is the functional region for CLV3, and that it is proteolytically processed from the CLV3 precursor protein. But the mechanism and activity responsible for this processing is poorly understood. Here we extend analysis of an in vitro CLE processing activity and show that in vitro cleavage occurs at Arg70, exactly matching in vivo maturation. We provide evidence that related processing activities are present in multiple tissues and species. We show that efficient protease recognition can occur with as little as four residues upstream of the CLE domain, and that the conserved arginine at position +1 and conserved acidic residues at positions -2 and/or -3 are required for efficient cleavage. Finally, we provide evidence that the N-terminal processing enzyme is a secreted serine protease while C-terminal processing may occur via a progressive carboxypeptidase.
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA.
Jun
J
Ni
Yongfeng
Y
Guo
Huiyan
H
Jin
Jennifer
J
Hartsell
Steven E
SE
Clark
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001937:Brassica / Q000737:chemistry; D002268:Carboxypeptidases / Q000378:metabolism; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057057:Serine Proteases / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism
C118167:AT2G27250 protein, Arabidopsis; D029681:Arabidopsis Proteins; D010940:Plant Proteins; D011480:Protease Inhibitors; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D001120:Arginine; D005982:Glutathione Transferase; D002268:Carboxypeptidases; D057057:Serine Proteases; D007930:Leucine; C012211:ubenimex