Xyloglucan Xylosyltransferases XXT1, XXT2, and XXT5 and the Glucan Synthase CSLC4 Form Golgi-Localized Multiprotein Complexes    

Abstract

Xyloglucan is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants and has important structural and physiological functions in plant growth and development. In Arabidopsis (Arabidopsis thaliana), the 1,4-b-glucan synthase, Cellulose Synthase-Like C4 (CSLC4), and three xylosyltransferases, XXT1, XXT2, and XXT5, act in the Golgi to form the xylosylated glucan backbone during xyloglucan biosynthesis. However, the functional organization of these enzymes in the Golgi membrane is currently unknown. In this study, we used bimolecular fluorescence complementation and in vitro pull-down assays to investigate the supramolecular organization of the CSLC4, XXT1, XXT2, and XXT5 proteins in Arabidopsis protoplasts. Quantification of bimolecular fluorescence complementation fluorescence by flow cytometry allowed us to perform competition assays that demonstrated the high probability of protein-protein complex formation in vivo and revealed differences in the abilities of these proteins to form multiprotein complexes. Results of in vitro pull-down assays using recombinant proteins confirmed that the physical interactions among XXTs occur through their catalytic domains. Additionally, coimmunoprecipitation of XXT2YFP and XXT5HA proteins from Arabidopsis protoplasts indicated that while the formation of the XXT2-XXT2 homocomplex involves disulfide bonds, the formation of the XXT2-XXT5 heterocomplex does not involve covalent interactions. The combined data allow us to propose that the proteins involved in xyloglucan biosynthesis function in a multiprotein complex composed of at least two homocomplexes, CSLC4-CSLC4 and XXT2-XXT2, and three heterocomplexes, XXT2-XXT5, XXT1-XXT2, and XXT5-CSLC4.The major structural components of plant cell walls are polysaccharide networks composed of pectins, hemicelluloses, and cellulose. The precise arrangement and composition of these components exert important influences on plant growth and development. Moreover, understanding (and possibly manipulating) cell wall formation and structure is key for industrial applications such as the production of biofuels and biomaterials. In dicotyledons and nongraminaceous monocotyledons, xyloglucan (XyG) is the major hemicellulosic polysaccharide (Scheller and Ulvskov, 2010). XyG has a backbone made of b-1,4-linked glucosyl residues and is branched, with short side chains made of Xyl, Gal, and Fuc. For example, in Arabidopsis (Arabidopsis thaliana), the basic repeating XyG subunit is XXXG, which is composed of a b-1,4-glucan, where three of four glucosyl residues are linked to a-D-xylosyl residues at the O-6 position. These basic XXXG repeating subunits can be further substituted at the O-2 position of the xylosyl residues by either a b-D-galactosyl residue (L) or a dimer, a-L-fucosyl-(1,2)-b-D-galactosyl (F), forming XXLG, XLLG, and XLFG subunits, respectively (Fry et al., 1993).All polysaccharides in plants, except cellulose and callose, are assembled by Golgi membrane-bound glycan synthases and glycosyl...

Time-Resolved Fluorescence Imaging Reveals Differential Interactions ofN-Glycan Processing Enzymes across the Golgi Stack in Planta

“…The observed interactions were largely consistent, which shows that enzyme pairs can interact through their N-terminal CTS regions. This observation disagrees with the findings for several mammalian N-glycan processing enzymes (Hassinen et al, 2010) and some plant cell wall biosynthetic enzymes such as GAUT1/GAUT7 (Atmodjo et al, 2011), ARAD1/ARAD2 (Harholt et al, 2012), or XXT2/XXT5 (Chou et al, 2012), where the catalytic domain is the key determinant. However, we cannot exclude the possibility that the catalytic domain might play a role in a potential complex formation between medial-and trans-Golgi enzymes.…”

“…Only recently, Harholt et al (2012) reported that the two pectin biosynthetic enzymes ARABINAN DEFI-CIENT1 (ARAD1) and ARAD2 form homodimeric and heterodimeric complexes, which are also held together by disulfide bonds. Also, there is evidence that the three xylosyltransferases XXT1, XXT2, and XXT5 as well as the glucan synthase CELLULOSE SYNTHASE-LIKE C4, all involved in xyloglucan biosynthesis in the Golgi, assemble into several homocomplexes and heterocomplexes that are part of a higher order protein complex (Chou et al, 2012).…”

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage

“…The synthesis of xyloglucan, another hemicellulose, requires homocomplexes and heterocomplexes of CSLC4, a b-1,4-glucan synthase (Cocuron et al, 2007), and XXT proteins (Chou et al, 2012(Chou et al, , 2015. Since CSLA2 and MUCI10 are members of the same GT families as CSLC4 and XXTs, respectively, future studies should investigate if similar protein-protein interactions facilitate GGM synthesis.…”

AspWood: High-Spatial-Resolution Transcriptome Profiles Reveal Uncharacterized Modularity of Wood Formation in Populus tremula

“…We identified the Populus genes encoding glycosyl transferases involved in the biosynthesis of homogalacturonan (Atmodjo et al, 2011), RG-I (Harholt et al, 2006;Liwanag et al, 2012), RG-II (Egelund et al, 2006), and xyloglucan (Cavalier and Keegstra, 2006;Zabotina et al, 2008;Chou et al, 2012) (Supplemental Data Set 9). As expected, the majority of these genes were highly expressed in primary walled cambial and radially expanding tissues (sample cluster ii; Figure 5A).…”