Figure 4.

Ste14p methyltransferase activity cofractionates with ER membranes by subcellular fractionation. A total yeast lysate derived from SM2915 that expresses a chromosomal copy of STE14 was subjected to fractionation on a sucrose step gradient. Equivalent volumes from each fraction were assayed for methyltransferase activity, for various organellar enzyme activities, and for protein concentration. Two populations of membranes can be identified. Light membranes marked by vacuolar α-d-mannosidase (Ams1p) and trans-Golgi network Kex2p activities are distributed at the top of the gradient (with peaks at fractions 2–3); heavier membranes marked by NADPH cytochrome c reductase (ER) and vanadate sensitive ATPase activities (plasma membrane) are distributed near the bottom of the gradient (fractions 6–10) with the peak distribution of these two compartments offset by one fraction. Methyltransferase activity cofractionates with the heavy ER membrane population. The dashed vertical line represents an arbitrary division between light and heavy membranes.