Fig 2.

Determination of lysine residues required for ubiquitin-dependent Tat1 degradation. 3HA-Tat1 proteins with multiple K → R substitutions (A) or single or double K → R substitutions (B) were visualized by Western blotting using an anti-HA antibody. The plasmids corresponding to the AS numbers are listed in Table 2. Asterisks denote plasmids containing either the K29R or K31R substitution (*) or both the K29R and K31R substitutions (**). Arrowheads denote the higher-molecular-mass bands of 3HA-Tat1. (C) Wild-type cells expressing the 3HA-Tat1 proteins with single or multiple K → R substitutions were exposed to a pressure of 25 MPa for 3 h. Whole-cell extracts were prepared for Western blotting to detect the variant 3HA-Tat1 proteins. Adh1 was used as a loading control.