Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and i nserted into the wall via exocytosis. The most abundant pectic polysacchari de, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The s ubcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradient s. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reti culum, was detected in the Golgi fraction. Homogalacturonan methyltransfera se activity was not reduced by protease treatment of intact membranes or me mbranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was r educed by protease treatment of membranes permeabilized with 0.02% Triton X -100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and t he lack of inhibition of HGA-MT activity by protease-treatment of intact me mbranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi.