Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, to date, no Golgi SAM transporter has been identified in plants. In this work, we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knock-out of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesised polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, the NMR revealed the existence of pectin 'egg-box' structures in intact cell walls, and showed that their formation is enhanced by reduced methyl-esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters that import SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.All plant cells are enclosed by a network of cell wall polysaccharides. This network must be strong enough to resist internal pressures yet remain flexible to permit cell growth. The balance between these attributes is governed by the properties of the cell wall, which are determined by the structure, chemistry and the interactions of its constituent polysaccharides.With the exceptions of cellulose and callose, cell wall polysaccharides are synthesised and modified in the Golgi apparatus, where various enzymes catalyse the transfer of glycosyl, acetyl, and methyl moieties onto glycan acceptors from a range of donor substrate molecules. Many of these donor substrates need to cross the Golgi membrane barrier in order to reach the lumen of the organelle. To achieve this, the Golgi membrane contains several types of transporter proteins that import essential metabolites, such as nucleotide sugars, acetylation donors, and the methylation donor S-adenosyl methionine (SAM) 1,2 .Various residues can be methylated in plant cell wall polysaccharides: glucuronic acid (GlcA) of xylan and arabinogalactan proteins 3,4 , various sugars in the pectin rhamnogalacturonan II (RG-II) 5 , and galacturonic acid (GalA) in the pectin homogalacturonan (HG), the most abundantly methylated polysaccharide 6 . The degree of HG methylation is considered a major factor influencing the capacity of cells to expand [7][8][9] . The 'egg-box' model describes the capacity of HG with a low .
Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, to date, no Golgi SAM transporter has been identified in plants. In this work, we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knock-out of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesised polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, the NMR revealed the existence of pectin egg-box structures in intact cell walls, and showed that their formation is enhanced by reduced methyl-esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters that import SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.
ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIβ, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localisation. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376motif. Taken together, our results demonstrate that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins.
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