The Import of S-Adenosylmethionine into the Golgi Apparatus Is Required for the Methylation of Homogalacturonan

Abstract

S-adenosylmethionine (SAM) is the substrate used in the methylation of homogalacturonan (HGA) in the Golgi apparatus. SAM is synthesized in the cytosol, but it is not currently known how it is then transported into the Golgi. In this study, we find that HGA methyltransferase is present in Golgi-enriched fractions and that its catalytic domain faces the lumen of this organelle. This suggests that SAM must be imported into the Golgi. We performed uptake experiments using [methyl-14 C]SAM and found that SAM is incorporated into the Golgi vesicles, resulting in the methylation of polymers that are sensitive to pectinase and pectin methylesterase but not to proteases. To avoid detecting the transfer reaction, we also used [carboxyl-14 C]SAM, the uptake of which into Golgi vesicles was found to be sensitive to temperature, detergents, and osmotic changes, and to be saturable with a K m of 33 mM. Double-label uptake experiments using [methyl-3 H]SAM and [carboxyl-14 C]SAM also revealed a time-dependent increase in the 3 H to 14 C ratio, suggesting that upon transfer of the methyl group, the resulting S-adenosylhomocysteine is not accumulated in the Golgi. SAM incorporation was also found to be inhibited by S-adenosylhomocysteine, whereas UDP-GalA, UDP-GlcA, and acetyl-CoA had no effect. DIDS, a compound that inhibits nucleotide sugar transporters, also had little effect upon SAM incorporation. Interestingly, the combination of UDP-GalA 1 acetyl-CoA or UDP-GlcA 1 acetyl-CoA produced a slight increase in the uptake of SAM. These results support the idea that a SAM transporter is required for HGA biosynthesis.

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells

“…3,6,8,[11][12][13][14][15] Nevertheless, in our study, the catalytic domain of GLMT1 seems to face the cytosolic side of the Golgi apparatus rather than the lumen. Thus, GLMT1 may not be involved in pectin biosynthesis.…”

“…14) QUA3 is a Golgi-localized type II membrane protein that has been predicted to function as a pectin MTase and that may be involved in the biosynthesis and modification of cell wall pectin. 8,15) In tobacco, homogalacturonan MTase activities have been detected in the Golgi fractions from cell suspensions. 6,11,12) Although GLMT1 has sequence similarity with QUA3 in its MTase domain, the closest homolog of GLMT1 in Arabidopsis is the At1g26850 protein, and the whole protein sequences are well conserved among these proteins.…”

“…Therefore, GLMT1 and possibly its orthologs may exhibit a methylation activity that shows distinct substrate specificity for several MTases having pectin-modifying properties. 3,8,14,15,27,28) A future search for the substrate of GLMT1 will be needed to elucidate the cellular function of this protein.…”

“…2,3) In plants, methyltransferase activities have been detected in the Golgi fractions from tomato, flax, mung beans, and tobacco, and these activities have been shown to play an essential role in pectin biosynthesis. [4][5][6][7][8][9][10][11][12] OSU1/QUA2/TSD2, which is a Golgi-localized predicted type II membrane protein acting as a pectin MTase, is a critical modulator of the carbon and nitrogen nutrient balance response and is involved in cell adhesion and the coordination of plant development. 3,13,14) QUA3 is a homogalacturonan MTase and is a Golgi-localized type II membrane protein in Arabidopsis.…”

Methylated glycans as conserved targets of animal and fungal innate defense

“…The respective gene was therefore named samt-1. Esterification of glucuronic acid residues with methyl groups in plant pectins is another SAM-dependent process taking place in the Golgi (41). Indeed, homologs of SAMT-1 are found encoded in plant genomes and may represent essential players for this common modification.…”