The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicat ed in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (B233 Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This wa s due to loss of their receptors from the cell surface The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate di stribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant S KD1-localized compartments. Degradation of epidermal growth factor was inhi bited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containin g TfR and endocytosed horseradish peroxidase. The early endosome antigen EE A1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane t raffic through them.