Effects of heterologous human tau protein expression in yeast models of proteotoxic stress response

Abstract

Background: The primary histological characteristic of Alzheimer's disease is the presence of neurofibrillary tangles, which are large aggregates of tau protein. Aging is the primary risk factor for the development of Alzheimer's disease, however, the underlying causes of tau protein aggregation and toxicity are unclear.

Aims: Here we investigated tau aggregation and toxicity under the conditions of compromised protein homeostasis.

Methods: We used heterologous expression of human tau protein in the unicellular eukaryote yeast Saccharomyces cerevisiae with evolutionarily conserved protein quality control pathways and examined tau-dependent toxicity and aggregation using growth assays, fluorescence microscopy, and a split luciferase-based reporter NanoBiT.

Results: Tau protein expressed in yeast under mild proteotoxic stress, or in mutants with impaired pathways for proteotoxic stress response, did not lead to synthetic toxicity or the formation of obvious aggregates. Chronologically old cells also did not develop observable tau aggregates. Our examination of tau oligomerization in living cells using NanoBiT reporter suggests that tau does not form significant levels of oligomers under basal conditions or under mild proteotoxic stress.

Conclusion: Together our data suggest that human tau protein does not represent a major burden to the protein quality control system in yeast cells.

Keywords: chronological aging; proteasome; protein aggregation; protein homeostasis; protein interaction assay; protein quality control; toxicity; yeast.

FIGURE 1

Effect of heterologous human tau protein expression on yeast cell viability in control and rpn4Δ and UPR mutants. (A) Diagrams of PCUP1‐tau and PGAL1‐tau constructs for inducible tau protein expression from CUP1 or GAL1 gene promoters (PCUP1, PGAL) via copper ion and galactose induction, respectively. (B, C) Inducible human tau protein expression in yeast cells was examined using immunoblots. Yeast strain BY4741 transformed with an empty vector (pXP731) or PCUP1‐tau (pKZ10) (B) or PGAL1‐tau (pKZ12) (C) were grown to the logarithmic growth phase and incubated for 2 h in the presence of 400 μM CuSO₄ (B) or galactose‐based medium (C), as indicated. The total cell lysate was immunoblotted with an anti‐tau antibody (Tau5). As a loading control, Bio‐Rad stain‐free technology (B) or Pgk1 (C) were used. Tau protein is only expressed at a significant level when cells are stimulated with CuSO₄ and galactose. In the absence of CuSO₄, cells with PCUP1‐tau show low tau expression. (D–I) Cell viability tests based on cell growth. The cells were grown to the logarithmic growth phase, and the culture was inoculated onto a solid medium in decimal dilutions. (D, E) Strains from (B) and (C) were inoculated onto solid media with and without 400 μM CuSO₄ (D) or galactose or glucose‐based media (C), respectively. After 3 days of incubation, no growth delays of cells grown under inducible conditions for tau expression were detected by dot spot. (F) Strain as in (C) was inoculated onto a galactose or glucose‐based solid medium and incubated at 30°C (optimal growth temperature) or 37°C (mild heat shock). After 3 days of incubation at 37°C, no growth delay of cells grown under inducible conditions for tau expression (+Gal) was detected by dot spot. (G) On galactose or glucose‐based solid media, the yeast deletion mutant strain rpn4Δ was inoculated after transformation with an empty vector (pXP722) or PGAL1‐tau (pKZ12). After 3 days of incubation, no growth delays of rpn4Δ cells grown under inducible conditions for tau expression (+Gal) were observed by the dot spot. (H, I) Yeast deletion mutant strains ire1∆ and hac1∆ transformed with an empty vector (pXP722) or PGAL1‐tau (pKZ12) were grown in the same manner as in (F). After 3 days of incubation, no growth delays were observed in ire1∆ (H) and hac1∆ (I) mutants grown under inducible conditions for tau expression (+Gal) as measured by the dot spot.

FIGURE 2

Human tau protein localization in yeast cells under proteotoxic stress and in chronologically aged yeast cells. (A–E) Localization of tau‐sfGFP in cells with exponential growth. (A) Schematic presentation of the PTEF1‐tau‐sfGFP construct (pKZ24). (B) Wild‐type cells (BY4741) transformed with pKZ24 displayed diffuse sfGFP signal when grown under standard conditions and after 2 h of incubation under mild heat stress (37°C) or (C) in the presence of 1 mM L‐azetidine‐carboxylic acid (D). (E) A pKZ24‐transformed rpn4Δ mutant strain exhibited a diffuse sfGFP signal. (F) Localization of NeonGreen‐tau in chronologically aged wild‐type cells. Schematic presentation of the PPIR3‐NeonGreen‐tau construct (pKZ51) is shown. NeonGreen‐tau is diffusely localized in cells from 3‐days old cultures of the wild‐type strain (BY4741 transformed with pKZ51). Scale bar = 10 μm.

FIGURE 3

Tau‐NanoBiT reporter for tau–tau interaction detection. (A) Schematic presentation of the NanoBiT (left) and tau‐NanoBiT (right) reporter constructs. Details can be found in the main text. Indicated are the small subunit (SmBiT), large subunit (LgBiT), TDH3 gene promoter (PTDH3), HA, and V5 epitope tags. (B) Immunoblot analysis of yeast tau‐NanoBiT protein expression. The yeast wild‐type strain (BY4741) was transformed with plasmid pRS316 (empty vector), plasmid pKZ04 (Tau‐SmBiT), plasmid pKZ06 (Tau‐LgBiT), both plasmids pKZ05 and pKZ06 or plasmid pKZ08. (tau‐NanoBiT construct). Total protein lysates were analyzed by western blot using antibodies against tau (tau5) or against epitope tags (anti‐HA, anti‐V5). The last lane was imaged simultaneously from the same blot. (C) Measurement of the luminescence of living cells expressing NanoBiT constructs, as in (B). Only cells expressing tau fusions to both SmBiT and LgBiT exhibited detectable levels of luminescence. (D–G) Examining the specificity of tau‐tau interactions with the tau‐NanoBiT reporter. (D) Immunoblot analysis of the expression of the tau‐NanoBiT and SmBiT‐sfGFP‐NLS proteins in yeast cells. Wild‐type BY4741 strains expressing tau‐NanoBiT (pKZ08) or SmBiT‐sfGFP‐NLS (pCA1016) and tau‐LgBiT (pKZ06) were used to prepare total protein lysates. Lysates were analyzed by immunoblot using antibodies against tau (tau5) and epitope tags (HA and V5). There are three independent replicates for each sample. The data were analyzed using a t‐test with two tails. On the graphs, the signal from the immunoblot on the left is quantified. A schematic illustration of the tested construct SmBiT‐sfGFP‐NLS is shown. Details can be found in the main text. (E) Luminescence measurement of living cells expressing NanoBiT as described in (D). p = 0.1 was determined using a two‐sided Mann‐Whitney test on the data. (F) Immunoblot analysis of the expression of the tau‐NanoBiT and Tdh3‐LgBiT proteins in yeast cells. Total protein lysates from the wild‐type strain (BY4741) expressing tau‐NanoBiT (pKZ35) or TDH3‐LgBiT and Tau‐SmBiT (pKZ15 and pKZ05) were analyzed by western blot with antibodies against tau (tau5) and epitope tags (HA and V5). There are three independent replicates for each sample. The data were analyzed using a t‐test with two tails. On the graphs, the signal from the immunoblot on the left is quantified. Presented is a schematic illustration of the construct Tdh3‐LgBiT. Details can be found in the main text. (G) Luminescence measurement of living cells expressing NanoBiT as described in (F). The data were analyzed using the two‐sided Mann‐Whitney test; p > 0.9999. All error bars represent the mean ± SD.

FIGURE 4

Analysis of the activation of the tau‐NanoBiT reporter in yeast cells under proteotoxic stress and in the rpn4∆ mutant. (A, B) Measurement of the luminescence of living cells expressing the tau‐NanoBiT reporter (pKZ08). (A) Log‐growing wild‐type cells (BY4741) were grown at 30°C to an OD₆₀₀ of 1.0, and aliquots were incubated for 30 min at 30 or 37°C. After treatment, luminescence was measured at RT immediately. (B) The luminescence of log‐growing wild‐type (BY4741) and rpn4∆ mutant cells were measured at an OD₆₀₀ of 1.0. (C) Analysis of tau‐NanoBiT (pKZ08) expression by immunoblot in wild‐type (BY4741) and rpn4∆ cells. Total protein lysates were analyzed by western blot using antibodies against tau (tau5) or against epitope tags (anti‐HA, anti‐V5). There are three independent replicates for each sample. (D) The graphs display the signal quantification from the immunoblot on the left (C). All error bars represent the mean ± SD.