FIGURE 1.

NBD2* is an unstable protein. A, Ste6p is a yeast ATP-binding cassette transporter containing 12 transmembrane domains and two cytoplasmic nucleotide binding domains. A mutation, Q1249X, in the second nucleotide binding domain (NBD2) of Ste6p (termed Ste6p*) results in its recognition as an ERAD substrate. B, soluble NBD2 constructs were designed with an N-terminal triple HA tag as either full-length NBD2 or NBD2 containing the identical Q1249X mutation found in Ste6p* (NBD2*). C, all constructs were cloned into a multicopy (2μ) plasmid (pRS426) and were placed under the control of the yeast phosphoglycerate kinase (PGK1) promoter. D, a cycloheximide chase assay (as described under “Experimental Procedures”) was used to measure protein stability over time, and yeast lysates were blotted with anti-HA-HRP to detect NBD2 and NBD2*. G6PD was probed as a loading control.