FIGURE 4.

PDI associates with Mnl1p through noncovalent interactions as well as disulfide bonds. Membrane fractions were prepared from cells expressing WT Mnl1p-FLAG and a series of Cys → Ser Mnl1p-FLAG mutants and from those with a vector alone (vec) in the presence of 10 mm DTT. The membranes were washed, solubilized with Nonidet P-40 in the absence of DTT, and subjected to immunoprecipitation with the anti-FLAG antibody. Immunoprecipitated materials (IP) and 10% of the solubilized membranes prior to immunoprecipitation (Input 10%) were analyzed by nonreducing (–ME) or reducing (+ME) SDS-PAGE and immunoblotting with antibodies against Mnl1p (IB: Mnl1p) or PDI (IB: PDI). Reduced and oxidized forms of the Mnl1p-FLAG monomer are indicated as red and ox, respectively.