The Hsp31 chaperone family is involved in quality control of misfolded cytoplasmic ΔssCL*myc.

<p>(A) Sequence alignment of the three members of the Hsp31 chaperone family in the yeast W303 strain shows an extraordinary sequence homology of 99% between Hsp32 and Hsp33. When compared with Hsp31 they show a sequence homology of about 70%. (B) Growth tests of deletion strains of the E3-Ligase Ubr1, different chaperones of the Hsp31 family and the corresponding wild type (WT) strain, respectively. All strains defective in the <i>URA3</i> and <i>LEU2</i> genes, harbour a centromeric (CEN) plasmid with the <i>URA3</i> marker and expressing ΔssCL*myc under the control of the <i>PRC1 (CPY)</i> promoter. ΔssCL*myc is composed of the vacuolar protein carboxypeptidase yscY (CPY) harbouring the point mutation G255R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140363#pone.0140363.ref054" target="_blank">54</a>]. The protein is additionally deleted in the signal sequence required for transport into the endoplasmic reticulum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140363#pone.0140363.ref006" target="_blank">6</a>]. In order to perform growth tests it is fused to the enzyme β-isopropylmalate dehydrogenase (Leu2) necessary for leucine biosynthesis. For immunodetection it is C-terminally fused to a c-Myc tag. Cells were spotted in a five fold dilution series on solid selection medium lacking uracil and leucine and, as a control, solely uracil, respectively. (C) Growth tests of cells performed as described in the legend to Fig 1B, but expressing Leu2myc (β-isopropylmalate dehydrogenase C-terminally fused to a c-Myc tag) instead of the terminally misfolded substrate ΔssCL*myc. As ΔssCL*myc, the Leu2myc protein is also expressed under control of the <i>PRC1</i> promoter. (D) Growth tests of cells as described in the legend to Fig 1B, but using yeast strains transformed with a <i>URA3</i> marker-containing plasmid expressing the ERAD-L model substrate CTL*myc under control of the <i>GAL4</i> promoter. CTL*myc consists of the misfolded ER-lumenal carboxypeptidase yscY (CPY*) moiety, the last transmembrane domain of Pdr5, and the cytoplasmic β-isopropylmalate dehydrogenase C-terminally fused to a c-Myc tag (Leu2myc). In contrast to ΔssCL*myc, the CPY* moiety is localized in the ER lumen and therefore glycosylated (black hexagons). The Δ<i>der3/hrd1</i> strain defective in ubiquitination of ERAD-L substrates served as control.