Organization of the yeast Golgi complex into at least four funtionally distinct compartments

Pro-alpha-factor (pro-alpha f) is posttranslationally modified in the yeast Golgi complex by the addition of alpha 1,6-, alpha 1,2-, and alpha 1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the alpha 1,6- and alpha 1,3-mannosylation and Kex2p-dependent processing of pro-alpha f are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that alpha 1,2-mannosylation of pro-alpha f is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-alpha 1,6-D-mannanase (endoM) were used to quantitate the amount of each pro-alpha f intermediate during transport through the Golgi complex. We found that alpha 1,6-, alpha 1,2-, and alpha 1,3-mannose were sequentially added to pro-alpha f in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. Ln addition, most of the Golgi-modified pro-alpha f that accumulated in brefeldin A-treated cells received only alpha 1,6-mannosylattion as did similar to 50% of pro-alpha f transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an alpha 1,6-mannosyltransferase but lacks alpha 1,2-mannosyltransferase activity in vivo. We propose that the alpha 1,6-, alpha 1,2-, and alpha 1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial, trans, and trans-Golgi network of the yeast Golgi complex, respectively.