ADD66, a Gene Involved in the Endoplasmic Reticulum-associated Degradation of -1-Antitrypsin-Z in Yeast, Facilitates Proteasome Activity and Assembly

Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - Autophagy is robust in the add66Δstrain. ADD66, add66Δ, and atg14Δ strains were grown in rich media (YPD) or in synthetic complete medium lacking ammonium sulfate (starvation media, or "STV"). Protein extracts were prepared from each strain and under each condition and were resolved by SDS-PAGE and probed with an antiserum that recognizes the precursor (pre) and mature (m) forms of Ape1p. Anti-Sec61p antiserum was used as a loading control.
• Supplemental Figure 2 - Expression of Add66p-myc from its endogenous promoter complements the chymotrypsin-like activity defect in add66Δ yeast. The chymotrypsin-like (CTL), trypsin-like (TL) and peptidylglutamyl peptide hydrolyzing (PGPH) activities in extracts prepared from ADD66 (square) or add66Δ (triangle) strains containing either a vector control or an Add66p-myc expression vector, or purified 26S proteasome (circles, last column), were determined using a fluorogenic substrate, as described in the Materials and Methods. A total of 100 µg of cytosol and 0.5 µg of 26S proteasome was used in each reaction. Fluorescence was measured on a spectrofluorometer (emission at 380 nm and excitation at 436 nm) at the indicated time points treated with DMSO (closed symbols) or inhibitor (open symbols; CTL: MG132, TL: leupeptin, PGPH: MG-132). The relative activities were normalizing to the fluorescent signals for wild-type levels (first two columns) or the DMSO-treated 26S proteasome (last column) at 180 min, which were set at 100%.
• Supplemental Figure 3 - The levels of 26S proteasome subunits are unaltered in the add66Δ strain. (A) Proteins in clarified cytosol from ADD66 (black bar) and add66Δ (white bar) yeast were resolved by SDS-PAGE and western blot analyses were performed to detect the levels of 20S subunits, a component of the 19S particle (Cim5p), Sse1p, and Hsp82p. Relative protein levels were determined by quantifying the intensities of the various proteins, and then normalizing these values to the levels measured in the isogenic wild type strain. Data represents the means of 8 independent experiments, +/- SD. (B) A representative western blot used to amass the data in part A.
• Supplemental Figure 4 - The relative affinities of the subunits that mediate the CTL activity for specific inhibitors are not altered in extracts prepared from add66Δ yeast. The CTL activities in extracts prepared from ADD66 (open circles) and add66Δ (closed circles) strains were determined (as described in the Materials and Methods) at the indicated concentration of (A) epoxomicin and (B) MG-132. The relative activity was determined by normalizing the fluorescent signals to wild-type levels in reactions lacking inhibitor. The structures of the inhibitors are shown, and the data represents the means of 4 independent experiments, +/- SD.
• Supplemental Figure 5 - Sequence alignment of amino acid residues 17-124 of human PAC2 and residues 4-121 of yeast Add66p. Sequence identities are shown in black, and conservative substitutions are indicated in gray. Gaps are indicated by a dash.
• Supplemental Figure 6 - Fractionation of proteasomes in lysates prepared after Add66p-myc over-expression in wild type and add66 yeast. Cell extracts were prepared from an ADD66 and add66Δ strain containing a plasmid engineered for the constitutive, strong over-expression of Add66p-myc. A total of 5 mg of protein were then resolved on a linear glycerol gradient (4-25%) and fractions were collected. Proteins in every other fraction were examined for the presence of 20S subunits, a component of the 19S subunit (Cim5p), and Add66p-myc by western blot analysis. The migrations of molecular mass markers, which were analyzed in parallel, are indicated below the gel.
• Supplemental Figure 7 - Over expression of A1PiZ in add66Δ yeast can result in lethality. Ten-fold serial dilutions of ADD66 and add66Δ transformed with either control plasmids or plasmids expressing A1PiM or A1PiZ (all of which were URA-marked) under the transcriptional control of a galactose inducible promoter were grown at 30°C for 48 h on YPD, SC-URA+GLC, or SC-URA+GAL (left half of figure). The growth defect in the add66Δ strain expressing A1PiZ in the presence of galactose was seen in ~15% of transformants after 50 individual transformants were examined (one transformant in which this phenomenon was observed is denoted by an asterisk, and a transformant in which growth was unaffected is denoted by a double-dagger). To establish that the lethal phenotype was not because the cells had become auxotrophic for galactose utilization (i.e., Gal-), the following protocol was followed: add66Δ strains transformed with either a control plasmid or a plasmid containing A1PiM or A1PiZ under the transcriptional control of a galactose inducible promoter were re-plated three times in the presence of 5FOA (in order to select for cells lacking the expression vector). Next, the resulting cultures (right half of figure) were serial diluted on 5FOA (to confirm selection for yeast lacking vector), SC-URA+GAL (to confirm that the yeast did indeed lack the URA-marked vector), or SC-URA+GAL (to confirm the Gal+ phenotype).