Figure 1.

P-type ATPases Dnf1p and Dnf2p reside in the plasma membrane. (A) Yeast strains expressing HA-tagged Drs2p, Neo1p, Dnf1p, Dnf2p, or Dnf3p were lysed, and cellular membranes were fractionated on a sucrose step gradient. Gradient fractions were assayed by enzyme activity for Kex2p or immunoblotted using antibodies against the HA-epitope or several organellar markers. Fractionation profiles of markers shown are from the gradient containing membranes of cells expressing HA-tagged Dnf1p, but were determined for each gradient individually, giving similar results. PM, plasma membrane; IMM, inner mitochondrial membrane; ER, endoplasmic reticulum; LE, late endosomes; EE, early endosomes; LG, late Golgi. (B) Fluorescence microscopy of cells stained with a rat monoclonal anti-HA antibody. Arrows indicate regions of heavily labeled plasma membrane. Bar, 10 μm.