Ubr1-dependency of degradation of the luciferase substrates

The two generated plasmids encoding the two model substrates LucLeu2myc and LucDMLeu2myc are supposed to be localized in the yeast cytosol because of the absence of the C-terminal sequence motif for peroxisomal import (SKL). To verify the possibility of usage of both substrates in studying cytoplasmic protein quality control the influence of the ubiquitin ligase Ubr1 on the degradation of LucLeu2myc and LucLeu2DMmyc was examined

ColE1 origin 1670..2352 AmpR 913..1572

M13-rev 2720..2740 T3 2758..2777

GAL1 Promoter 2845..3508 4173 SphI (1)

LacZ alpha 6663..6731

pLUCLEU24MYC 8531 bp

CEN-ARS pRS 583..73

LacO 2692..2714 URA3 7240..8346

T7 6576..6549 F1 ori 6749..7055 M13-fwd 6592..6575

4MYC 6251..6450

LEU2 5161..6250

luciferase ORF 3514..5154

(Fig.3.11). The corresponding strains were either grown overnight in glucose- or in galactose-containing medium before cycloheximide chase experiments were performed (Fig. 3.11).

Using glucose medium only small amounts of the substrates are expressed because of the glucose-repressible GAL1 promoter. To check whether overexpression of substrate causes alterations in the degradation kinetics the strains were also grown overnight in galactose-containing medium causing overexpression of corresponding substrates.

Figure 3.11: Both the substrates LucLeu2myc and LucDMLeu2myc are degraded in a Ubr1-dependent manner when expressed at a low level. Yeast strains are transformed with plasmids either expressing the substrate LucLeu2myc or LucDMLeu2myc both under control of the GAL1 promoter. Glucose-containing growth medium was used for low expression of the model substrates (upper panel) or galactose-containing medium for high substrate expression (lower panel). Samples were taken at the indicated time points after addition of cycloheximide. PGK served as loading control.

Low expression of the substrates LucLeu2myc and LucDMLeu2myc is caused by the leakiness of the GAL1 promoter in glucose-containing medium. The degradation of both

substrates in the wild type strain is visible (Fig. 3.11 upper panel). The destabilized version LucDMLeu2myc is degraded more rapidly. Absence of the cytosolic E3 ligase Ubr1 causes a dramatic stabilization of both substrates showing its involvement in the degradation process.

Growth of the used yeast strains in galactose-containing medium causes massive expression of the two substrates. Even in the wild type strain no degradation can be observed for both substrates in the measured time period (Fig. 3.11 lower panel).

3.2.3 Detection of the influence of the Hsp70 chaperone Ssa1 on the protein quality control of the substrates LucLeu2myc and

LucDMLeu2myc using luciferase assays

An advantage of the substrates LucLeu2myc and LucDMLeu2myc is the easy measurement of their steady state levels via detection of luciferase activity. Luciferase converts the substrate luciferin into oxyluciferin under emission of chemiluminescence.

Chemiluminescence can be easily detected by photometric measurements. The involvement of the Hsp70 system in quality control of the two model substrates was examined using luciferase activity measurements. Yeast strains defective in the Hsp70 system and expressing either LucLeu2myc or LucDMLeu2myc were harvested either in exponential or in stationary growth phase and the cells were lysed for subsequent luciferase assays. The strains harbouring the ssa1-45^ts allele were shifted to 37 °C for 1h prior to harvesting and lysis. As control the strains were grown at 30 °C, conditions where the ssa1-45^ts allele produces mainly active Ssa1 protein. The strain harbouring the wild type SSA1 gene served as control strain for the measurements (Fig. 3.12).

Figure 3.12: In exponential phase loss of Ssa1 function dramatically decreases luciferase activity of the substrate-expressing strains. Both yeast strains harbouring the wild type SSA1 gene and the temperature-sensitive ssa1-45^ts allele were transformed either with the plasmids pIA14 or pIA15 coding for the substrates LucLeu2myc (Luc), LucLeu2DMmyc (LucDM) or the corresponding empty plasmid pRS316 (pRS). Cells were harvested in exponential growth phase at an OD₆₀₀ of 1.0 prior to cell lysis and luciferase measurements. The temperature-sensitive ssa1-45^ts strain was shifted to 37 °C prior to harvesting. The relative light units (RLU) were normalized to the total protein concentration of each cell lysate (specific activity). Plotted data represent the mean of three luciferase measurements. Error bars indicate the standard deviation of the mean.

Under both permissive (30 °C) and restrictive (37 °C) conditions lysates of both yeast strains expressing the luciferase-containing substrates exhibit luciferase activity (Fig. 3.12). Strains expressing the destabilized version of luciferase (LucDM) always show lower activities. As expected, control strains transformed with empty plasmid (pRS) do not show any luciferase

activity. The activity of lysates obtained from ssa1-45^ts strains at 30 °C show higher activities than lysates of SSA1 wild type strains both expressing the luciferase substrates (Fig. 3.12 upper panel). At restrictive temperature the lysates of the ssa1-45^ts strain show a dramatically decreased luciferase activity whereas in the wild type SSA1 situation the activities only moderately decrease after shift to 37 °C (Fig. 3.12 lower panel).

Next, the same assays were performed but using cultures grown for three days at 30 °C. One part of the cultures were shifted to 37 °C for 1 hour prior to lysis and luciferase assays (Fig.

3.13).

Figure 3.13: In stationary phase the mutated Ssa1 protein expressed from the ssa1-45^ts allele even at permissive temperature cannot retain luciferase activity. The same yeast strains and plasmids were used as described in the legends to Fig.3.12. The cells were harvested after three days of growth prior to lysis and luciferase measurements. The temperature-sensitive ssa1-45^ts strain was shifted to 37 °C prior to harvesting. The relative light units (RLU) were normalized to the total protein concentration of each cell lysate. Plotted data represent the mean of three luciferase measurements. Error bars indicate the standard deviation of the mean.

When grown to stationary phase the lysates of strains harbouring the temperature-sensitive ssa1-45^ts allele and expressing the luciferase-containing substrates do not exhibit any luciferase activity even at 30 °C. Only for the wild type SSA1 strains expressing the luciferase substrates luciferase activity can be detected which decreased after shift to 37 °C in a similar modest way like in exponential phase (Fig. 3.13). As mentioned previously in chapter 3.1.3, Ssa1 and Ssa2 are only expressed during exponential growth until diauxic shift. In contrast, Ssa3 is not detectable under exponential conditions but strongly expressed in stationary phase (Hasin et al, 2014; Werner-Washburne et al, 1989). This difference in expression of the Ssa chaperones might also explain the results in the stationary growth phase seen in Fig. 3.13 because the used strains are deleted in the SSA3 gene.

3.3 San1-dependency of degradation of cytoplasmic misfolded