Figure 4.

Localization and oligomerization of the Mnn1p mutants. (A) Immunofluorescence micrographs of Δmnn1 yeast containing CEN plasmids expressing myc-tagged Mnn1p, no protein (control), or the indicated myc-tagged Mnn1p mutants, analyzed with the anti-myc monoclonal antibody 9E10. For I434A, cells were double labeled with rabbit anti-myc and an ER marker (anti-HDEL monoclonal antibody 2E7). The expression level of all constructs varied somewhat between cells, presumably reflecting variations in CEN plasmid copy number, or the known cell cycle regulation of MNN1 (23). However, the intracellular distributions of the proteins were unaffected by expression level. I434A was also expressed in a strain in which the endogenous Mnn1p was HA tagged, and the cells were double labeled with anti-myc and anti-HA. (B) Multimerization of Mnn1p. Anti-SV5 tag protein blots of total protein, or of anti-myc immunoprecipitates, from yeast with the genomic copy of MNN1 tagged with SV5, and with CEN plasmids expressing no protein, or myc-tagged Mnn1p as wild type or D430A. The SV5-tagged Mnn1p is precipitated by the anti-myc tag only when a myc-tagged Mnn1p is present, and this interaction is not affected by the D430A mutation.