Xyloglucan xylosyltransferases XXT1, XXT2, and XXT5 and the glucan synthase CSLC4 form Golgi-localized multiprotein complexes.

Yi-Hsiang Chou,Gennady Pogorelko,Olga A Zabotina

doi:10.1104/pp.112.199356

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-β-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.

Highlights

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
bimolecular fluorescence complementation (BiFC) constructs for each pair of xylosyltransferases, XXT1-XXT2, XXT1-XXT5, and XXT2-XXT5, were transiently coexpressed in Arabidopsis protoplasts using the polyethylene glycol (PEG) method (Jin et al, 2001), and the signal from reconstituted yellow fluorescent protein (YFP) was examined by fluorescence microscopy
Recent reverse-genetic studies indicated that three xylosyltransferases, XXT1, XXT2, and XXT5, play different roles in XyG biosynthesis (Cavalier et al, 2008; Zabotina et al, 2008), and the presence of all three proteins is essential for the formation of the wild-type XyG (Zabotina et al, 2012)

Summary

Introduction

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. Most glycosyltransferases are localized in the Golgi membrane and have a type II membrane protein topology (Perrin et al, 2001), with a short N-terminal fragment most likely protruding into the cytosol, one helical transmembrane domain, and a catalytic domain containing a DXD motif, which is attached to a flexible stem region; both the catalytic domain and the stem region are localized in the Golgi lumen (Albersheim et al, 2010) These glycosyltransferases are highly specific; it is postulated that a distinct enzyme is required to create each type of linkage (Keegstra and Raikhel, 2001). Stolz and Munro (2002) demonstrated that several mannosyltransferases involved in cell wall mannan synthesis form two types of protein complexes, M-Pol I and II

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