Identification and characterization of the Arabidopsis GAUT1:GAUT7 pectin homogalacturonan:galacturonosyltransferase complex
Atmodjo, Melani Agustina
MetadataShow full item record
Pectin is a family of structurally complex polysaccharides defined by the presence of α-1,4-linked galacturonic acid (GalA) residues. It is present in all plant cell walls and contributes substantially to normal plant growth and development. Prior research has elucidated the intricate structure of pectin, yet a detailed understanding of pectin biosynthesis remains elusive. The membrane-bound Arabidopsis GALACTURONOSYLTRANSFERASE 1 (GAUT1) catalyzes the biosynthesis of homogalacturonan (HG), a linear homopolymer of α-1,4-linked GalA that makes up ~65% of total pectin. GAUT1 is an α-1,4-galacturonosyltransferase (GalAT) that transfers GalA from UDP-GalA onto HG acceptors to synthesize HG oligomers and polymers. This study presents the identification of an Arabidopsis HG:GalAT complex composed of GAUT1 and its homolog GAUT7, both of which are Golgi-localized, type II transmembrane proteins. Co-immunoprecipitation using anti-GAUT1 and anti-GAUT7 polyclonal antibodies and bimolecular fluorescent complementation demonstrated protein-protein interaction between GAUT1 and GAUT7. Transcript analyses indicate GAUT1 and GAUT7 co-expression in most tissues and developmental stages of Arabidopsis. Upon non-reducing SDS-PAGE, both GAUT1 and GAUT7 resolve at ~185 kDa, a size larger than their monomeric forms (~60 kDa and ~75 kDa, respectively) observed by reducing SDS-PAGE, thus indicating disulfide bond involvement in GAUT1:GAUT7 complex formation. Proteomics analyses identified GAUT1 and GAUT7 as the only components of the non-reducing SDS-PAGE-stable GAUT1:GAUT7 complex. The observed size of monomeric Arabidopsis GAUT1 (~60 kDa) is smaller than its predicted mass (77.4 kDa). N-terminal sequencing of GAUT1, mass spectrometry protein sequence coverage, and primary structure-dependent GAUT1 reactivity against a series of anti-GAUT1 antibodies, all suggest post-translational cleavage of Arabidopsis GAUT1 between Met167 and Arg168, resulting in a mature GAUT1 with no transmembrane domain. Transient expression of several GFP fusion constructs of GAUT1 in N. benthamiana leaves, in the presence or absence of GAUT7, showed a GAUT7-dependent Golgi accumulation of GAUT1-GFP. It is concluded that Golgi retention of GAUT1 is due to the formation of a complex with GAUT7. Models of the GAUT1:GAUT7 HG:GalAT complex, with GAUT1 as the catalytic subunit and GAUT7 as the membrane anchor, are proposed. Twelve additional proteins were also identified by proteomic analyses and proposed as GAUT1:GAUT7 complex putative interacting proteins.