Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex

Abstract

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

Fig. 1.

GAUT1 and GAUT7 interact in a protein complex. (A) Coimmunoprecipitation of GAUT1 and GAUT7. Anti-GAUT1 and anti-GAUT7 antibody-immunoadsorbed proteins from the Arabidopsis SP fraction separated by SDS/PAGE and immunoblotted with anti-GAUT1 or anti-GAUT7 sera. The experiment was done thrice with similar results. IgG, IgG heavy chain detected by secondary antibody; IP, immunoprecipitation; M, molecular mass protein marker. (B–H) BiFC analysis of GAUT1 and GAUT7. Constructs were transiently coexpressed in N. benthamiana leaves, with BiFC of ARAD1 as control (28). YFP signals are in yellow. Individual expression of each construct gave no YFP signal. Results were verified by three independent experiments (except ARAD1 negative control, which was done twice). (I–M) Arabidopsis GAUT1 and GAUT7 promoter::GUS construct expression in whole (I) and developing lateral roots (J) of 7-d-old seedlings, and in flowers (K), stems (L), and tap roots (M) of mature 6- to 8-wk-old plants. Similar results were observed in multiple T2 generation plants from at least five independent T1 lines. Ca, cambium; Co, cortex; Ep, epidermis; Mx, metaxylem; Ph, phloem; Px, protoxylem (L) or primary xylem (M); Sx, secondary xylem; VC, vascular cambium. Boxed areas in left panels of L are shown at higher magnification on the right panels.

Fig. 2.

The GAUT1:GAUT7 GalAT complex is selective for HG substrate and held together by covalent and noncovalent interactions. (A) GalAT activities of the Arabidopsis SP fraction and of anti-GAUT1– and anti-GAUT7–immunoprecipitated GAUT1:GAUT7 complex were tested at 0.1 and 1 μM pectic acceptors: OGA DP 7–23; RG-I oligomers DP 6–26 with either rhamnose (RG-I-R) or GalA (RG-I-G) at the nonreducing ends; and RG-II monomer. Data are mean ± SD (n = 3). (B) GAUT1 and GAUT7 resolve at higher masses in nonreducing SDS/PAGE. The Arabidopsis SP fraction separated by SDS/PAGE in the presence or absence of 25 mM DTT and analyzed by immunoblotting with anti-GAUT1 or anti-GAUT7 sera. Protein bands common to GAUT1 and GAUT7 are estimated at ∼185 kDa (noted as GAUT1:GAUT7 core complex). Arrowheads indicate additional GAUT1 or GAUT7 HMW protein bands. (C) Coimmunoprecipitation of GAUT1 and GAUT7 is abolished only in the presence of both denaturing and reducing agents. The Arabidopsis SP fraction was preincubated for 30 min under denaturing [0.05% (vol/vol) Nonidet P-40, 0.0125% (wt/vol) deoxycholate, 0.5% (wt/vol) SDS], reducing (50 mM DTT), or both denaturing and reducing conditions before immunoprecipitation using anti-GAUT7 antibody and subsequent Western analysis.

Fig. 3.

Golgi retention of cleaved GAUT1 relies on the presence of GAUT7. (A–F) Transient coexpression of GAUT1-GFP and GAUT7-YFP in N. benthamiana leaves. (F, Inset) Mean pixel intensity is plotted from each Golgi in the GFP versus YFP channels (detected by sequential scanning), showing Golgi signals in GAUT7-YFP individual expression experiments (◇) and GAUT1-GFP/GAUT7-YFP coexpression experiments (◆). The Inset reveals that GFP signals detected upon GAUT1-GFP/GAUT7-YFP coexpression (D) are due to the Golgi accumulation of GAUT1-GFP in the presence of GAUT7-YFP, and not to background signal from GAUT7-YFP (B). Results were verified in at least three independent experiments. (G–M) Transient expression of C-terminally truncated GAUT1-GFP fusion constructs (G) in the absence (H–J) or presence (K–M) of GAUT7. GAUT1-GFP, full-length GAUT1 fused to GFP; GAUT1(100)-GFP, first 100 aa of GAUT1 fused to GFP; GAUT1(291)-GFP, first 291 aa of GAUT1 fused to GFP.

Fig. 4.

A model of the Arabidopsis GAUT1:GAUT7 core complex.