The polyglutamine protein ataxin-3 enables normal growth under heat shock conditions in the methylotrophic yeast Pichia pastoris

Abstract

The protein ataxin-3 carries a polyglutamine stretch close to the C-terminus that triggers a neurodegenerative disease in humans when its length exceeds a critical threshold. A role as a transcriptional regulator but also as a ubiquitin hydrolase has been proposed for this protein. Here, we report that, when expressed in the yeast Pichia pastoris, full-length ataxin-3 enabled almost normal growth at 37 °C, well above the physiological optimum of 30 °C. The N-terminal Josephin domain (JD) was also effective but significantly less, whereas catalytically inactive JD was completely ineffective. Based on MudPIT proteomic analysis, we observed that the strain expressing full-length, functional ataxin-3 displayed persistent upregulation of enzymes involved in mitochondrial energy metabolism during growth at 37 °C compared with the strain transformed with the empty vector. Concurrently, in the transformed strain intracellular ATP levels at 37 °C were even higher than normal ones at 30 °C. Elevated ATP was also paralleled by upregulation of enzymes involved in both protein biosynthesis and biosynthetic pathways, as well as of several stress-induced proteins. A similar pattern was observed when comparing a strain expressing JD with another expressing its catalytically inactive counterpart. We suggest that such effects mostly result from mechanisms of transcriptional regulation.

Figure 1

Spotting assay of ATX3 variants. After an overnight growth at 30 °C, cultures were diluted as indicated, spotted onto YPD-Zeo plates and incubated for 3 days at 30 °C or 37 °C. For the heat shock assay (HS), the cultures were treated for 30 min at 48 °C, spotted as above, then incubated at 30 °C.

Figure 2

Growth curves of control strain (dots), or strains expressing JD (squares), JD-C14A (triangles) and ATX3Q26 (crosses) at 30 °C (A) or 37 °C (B). Cultures were grown in YPD-Zeo medium under constant shaking and growth profiles monitored. Error bars represent standard deviations and are derived from at least three independent replicates. In all cases, they are smaller than the symbols used.

Figure 3

Differential changes in P. pastoris proteome as determined by MudPIT analysis during the growth at 37 °C. Differential expression of the proteins was determined at the indicated growth times using the MAProMa comparison between the ATX3-trasformed versus the control P. pastoris strain, and JD-transformed versus JD-C14A-transformed strain, respectively. Each protein was identified by its Entry Uniprot (See Supplementary Table S4 for the corresponding Gene name and complete reference). For each growth time, a color code was assigned that represents the respective DAve value. The relevant chromatic scale (reported in figure) ranges from −2.00 to −0.40 (dark blue to white) and from +0.40 to +2.00 (white to dark red). Proteins were primarily grouped according to their molecular function and secondarily by their increasing GI Accession numbers. The complete list of the proteins presented was extracted from the differential lists in Supplementary Table 4. Only proteins with unambiguously assigned functions are included.

Figure 4

Differential changes in P. pastoris proteins related to mitochondrial energy metabolism during the growth at 37 °C. The differential expression was determined at 48 h growth time using the MAProMa comparison between the ATX3-trasformed- versus the control strain, and JD-transformed- versus JDC14A-transformed strain. Each protein was identified by its gene name (see Supplementary Table S5 for the complete reference) and represented as a node. Protein-protein interactions are indicated as grey edges. The color code of distinct nodes represents the DAve value and the relevant chromatic scale (reported in figure) ranges from −2.00 to 0 (dark blue to white) and from 0 to + 2.00 (white to dark red). The bold borders indicate DAve and DCI values that pass the filters: DAve ≥ l0.4 l and a DCI ≥ l400l. The proteins are grouped into four mitochondrial sub-pathways.

Figure 5

Determination of ATP levels at different growth times at 30 °C or 37 °C in strains expressing ATX3 variants. Cultures were grown at 30 °C (A) or 37 °C (B). At the indicated times samples were withdrawn and ATP assayed using the ENLITEN® ATP Assay System Bioluminescence Detection Kit. Data are expressed as nanomol of cells. Each experiments was performed in technical triplicate. Error bars represent standard deviations and are derived from at least three independent replicates. *p value < 0.01.