Identification of Ahp1p as a target of the urmylation pathway. (A) Lysates of urm1Δ cells (SY4139) and urm1Δ cells expressing His₆- and FLAG epitope-tagged Urm1p (SY4118) were subjected to affinity chromatography on Talon resin. Eluates from the Talon resin were applied to anti-FLAG agarose. The proteins eluted from the FLAG agarose were analyzed by SDS-PAGE and immunoblotting with polyclonal antibodies against Urm1p. The L bracket designates high-molecular-weight Urm1p-modified species, while the asterisk indicates the position of a 32-kDa Urm1p-modified species. (B) BLAST sequence analysis of five peptides (underlined) obtained from the MS-identified Ahp1p.

Urm1p coimmunoprecipitates with Ahp1p from whole-cell extracts. Exponential cultures of haploid strains SY4050 (wild type) carrying GST-HisX6 and SY3972 (wild type) carrying GST-HisX6-Ahp1p were lysed in the presence of 20 mM N-ethylmaleimide. The lysates were analyzed by SDS-PAGE and immunoblot analysis using affinity-purified polyclonal anti-Urm1p. The bands corresponding to Urm1p conjugated to Ahp1p are indicated.

A 32-kDa band disappears in ahp1Δ cells. Strains SY3358 (wild-type), SY3806 (urm1Δ), SY3805 (uba4Δ), and SY3815 (ahp1Δ) were grown to mid-log phase in YEPD at 30°C. The lysates were analyzed by SDS-PAGE and immunoblot analysis using affinity-purified polyclonal anti-Urm1p antibodies and monoclonal antibodies to Dpm1p (to confirm equal protein loading). The L bracket designates high-molecular-weight Urm1p-modified species, while the asterisk designates the position of a 32-kDa Urm1p-modified species.

Ahp1p functions in TOR signaling. (A) Strains SY3836 (gln3Δ), BY4741 (wild type [WT]), SY3839 (urm1Δ), SY3840 (uba4Δ), SY3845 (ure2Δ), and SY4121 (ahp1Δ) carrying pTOR2 (pML40) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/10) was performed starting with 10,000 cells. Cells were spotted onto either SD-LEU (left) or SD-LEU plus 25 nM rapamycin (right) and grown for 3 days at 30°C. (B) Strains SY3836 (gln3Δ), BY4741 (wild-type), SY3839 (urm1Δ), SY3840 (uba4Δ), SY3845 (ure2Δ), and SY4121 (ahp1Δ) carrying pTOR2-1 (pML41) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/10) was performed starting with 10,000 cells. Cells were spotted onto SD-LEU (left) or SD-LEU plus 25 nM rapamycin (right) and grown for 3 days at 30°C.

Sensitivities of the urmylation pathway mutants to oxidative stress. (A) Loss of the urmylation pathway does not result in sensitivity to t-BOOH. Strains BY4741 (wild type [WT]), SY4119 (ahp1Δ), SY3839 (urm1Δ), SY3840 (uba4Δ), SY4122 (ahp1Δ urm1Δ), SY4123 (ahp1Δ uba4Δ), and SY4124 (urm1Δ uba4Δ) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/5) was performed starting with 10,000 cells. Cells were spotted onto either YEPD plus 0.4 mM t-BOOH (left), YEPD plus 1 mM t-BOOH (middle), or 1.5 mM t-BOOH (right) and grown for 3 days at 30°C. (B) Loss of Ahp1p and the urmylation pathway results in diamide sensitivity. Strains BY4741 (wild type), SY4119 (ahp1Δ), SY3839 (urm1Δ), SY3840 (uba4Δ), SY4122 (ahp1Δ urm1Δ), SY4123 (ahp1Δ uba4Δ), and SY4124 (urm1IΔ uba4IΔ) were grown to mid-log phase in YEPD at 30°C. A serial dilution (1/5) was performed starting with 10,000 cells. Cells were spotted onto either YEPD (left), YEPD plus 1 mM diamide (middle), or 1.3 mM diamide (right) and grown for 3 days at 30°C.

The Ahp1p-Urm1p conjugate is affected by diamide and t-BOOH treatment. Strain SY3357 (wild type) was grown to mid-log phase in YEPD at 30°C. Cells were then grown in YEPD at 30°C in the presence of 1.5 mM diamide or 1.3 mM t-BOOH or without drug treatment for 4 h. The lysates were analyzed by SDS-PAGE and immunoblot analysis using affinity-purified polyclonal anti-Urm1p antibodies and monoclonal antibodies to Dpm1p.