Figure 2. Native start site and aggregation analysis of Mca1.

(A) Mca1₄₃₂ and Mca1₄₅₄ (N-terminal extension MGKMSLEVYLNYHQRRPTRFTI) were expressed from a copper-inducible promoter in the presence of 150 µM CuSO4 and endogenous Mca1 was expressed from its native promoter in a strain lacking the chromosomal MCA1 gene (Y103). Mca1 protein was detected using polyclonal anti-Mca1 antibodies and immunoblotting. Plasmids used in this assay: V294 (vector control  =  VC), V413 (Mca1₄₃₂), V414 (Mca1₄₅₄) and V415 (endogenous Mca1). (B) MCA1 ₄₃₂ and MCA1 ₄₅₄ (N-terminal extension MGKMSLEVYLNYHQRRPTRFTI) were expressed from a copper-inducible promoter and endogenous Mca1 was expressed from its native promoter in a mca1Δ strain (Y103). Soluble and aggregated proteins were separated by low-spin (18,000×g) and high-spin (100,000×g) centrifugation. Mca1 protein was detected using polyclonal anti-Mca1 antibodies (raised against Mca1₄₅₄) and immunoblotting. Rnq1 was detected using polyclonal anti-Rnq1 antibodies. (T) total lysate; (LS) low-spin supernatant fraction; (LP) low-spin pellet fraction; (HS) high-spin supernatant fraction; (HP) high-spin pellet fraction. Plasmids used in this assay: V294 (vector control  =  VC), V413 (Mca1₄₃₂), V414 (Mca1₄₅₄) and V415 (endogenous Mca1). Please note that the two smaller fragments of Mca1₄₅₄ seen in A (see text for details) were also present in B, albeit not shown in this section.