FIG 3.

GiMOMP35 is an outer mitosomal membrane protein. (A) Protease protection assay of high-speed pellets isolated from Giardia expressing HA-tagged GiMOMP35. After incubation with trypsin, the samples were immunolabeled with antibodies against the HA tag, the outer membrane protein GiTom40, and the matrix protein GiIscU. The sensitivity of GiMOMP35 to the protease indicates its outer membrane localization. The drawing shows the suggested topology of GiMOMP35. Cyt, cytosol; OM, outer mitosomal membrane. (B) Cells expressing HA-tagged GiMOMP35 were stained with an anti-HA tag antibody (green) and an anti-GL50803_9296 antibody (red). Nuclei were stained with DAPI (blue). In addition to exhibiting typical mitosomal morphology (B), approximately 50% of the observed trophozoites contained elongated tubular structures (C to E). These structures colocalized with GiTom40 (red) (C); however, only a small fraction exhibited costaining for GL50803_9296 (red) (D). The structures were devoid of the ER marker GiPDI2 (red) (E). These data indicate that the elongated tubular structures represent an enlarged outer mitosomal membrane. Under transmission electron microscopy, the structures appeared as organized membrane layers (F).