Figure 6.

Epitope tagging of Gga1p. (a) An HA tag was engineered onto the COOH terminus of Gga1p and expressed at either endogenous levels (HA[CEN]) or higher than normal levels (HA[2μ]). Cells were pulse-chased and total CPY was immunoprecipitated. The tagged construct gives full rescue at both endogenous and high levels. (b) Cells expressing HA-tagged Gga1p on a 2μ plasmid were metabolically labeled and then immunoprecipitated with anti-HA, together with cells expressing wild-type Gga1p (wt) as a control. The autoradiograph shows that the tagged construct is recognized by anti-HA. (c) Localization of the tagged Gga1p in a vps4 mutant. Cells were double labeled for HA-tagged Gga1p (top) and myc-tagged Vps10p (bottom). Vps10p is known to go to the class E compartment in a vps4 mutant; therefore, the colocalization of Gga1p with Vps10p indicates that it also goes to the class E compartment. The reason for the use of a class E mutant is that late Golgi, endosomal, and vacuolar proteins all accumulate in an exaggerated prevacuolar compartment, the class E compartment, making the membrane localization of tagged Gga1p much easier to see. Scale bar, 5 μm.