Figure 10.

Possible sites of action of Lec35 protein. A schematic representation of MPD-dependent mannosylation reactions in the ER membrane is shown. Lec35 protein (Lec35p) is required for mannosylation of at least three classes of acceptor substrate (LLO, GPI, tryptophan residues of specific proteins) represented as a black oval oriented at the lumenal side of the ER membrane. Reactions that do not require Lec35p directly are shown by solid arrows, whereas reactions that might involve Lec35p are indicated by dotted arrows. The experiments reported here show that the catalytic transfer of mannose from MPD to acceptor does not directly involve Lec35p, and previous work established that MPD synthesis did not require Lec35p. As explained in the INTRODUCTION, any proposed mechanism must have the potential to account for correction of the Lec35 phenotype by physical or chemical perturbation. (i) Lec35p might be involved on the cytoplasmic leaflet in movement or exchange of MPD from MPD synthase to the MPD flippase (indicated by paired trapezoids). (ii) Lec35p might be involved in MPD flippase function, perhaps as the flippase itself, a regulatory subunit, or a regulator of its expression. As discussed in the text, it appears unlikely that Lec35p is involved in the function of the flippase-like activity assayed by MPC transport. However, there still remains a possibility of other uncharacterized MPD flippase activities (ii*) that require Lec35p. (iii) Lec35p might be involved on the lumenal leaflet in movement or exchange of MPD from the flippase to the various mannosyltransferases. (iv) Lec35p might be necessary to prevent missorting of MPD to an enzymatically inactive domain of the ER membrane, or to another organelle.