Influence of the Hsp31 chaperone family on the quality control of the substrates LucLeu2myc

In cycloheximide chase experiments it has been shown that the luciferase-based model substrates LucLeu2myc and LucDMLeu2myc are degraded in a Ubr1-dependent manner (Fig.

3.11). In order to confirm these results growth tests were performed making use of the auxotrophic marker Leu2 of the two luciferase-based substrates. In addition, growth tests were performed using strains lacking the members of the Hsp31 chaperone family in order to analyse the influence of the chaperones on the steady state levels of the two luciferase-containing substrates (Fig. 3.41).

Figure 3.41: The steady state levels of LucLeu2myc and LucDMLeu2myc are influenced by the E3 ligase Ubr1 and the Hsp31 chaperone family. Growth tests were performed with yeast strains transformed with either the plasmid pIA14 or pIA15 coding for the substrates LucLeu2myc or LucDMLeu2myc respectively. Substrate levels were monitored using medium lacking leucine whereas medium only lacking uracil monitors the presence of the used plasmids. Low expression was achieved in glucose-containing medium (upper panel) whereas galactose-containing medium caused high induction of substrate expression due to the GAL1 promoter (lower panel).

Low expression of the two substrates LucLeu2myc and LucDMLeu2myc upon use of glucose-containing medium results in visible growth differences of the used yeast strains on plates lacking leucine. The wild type strains exhibit the weakest growth on medium lacking

leucine corresponding to the lowest substrate steady state levels (Fig. 3.41 upper panel).

Similar to the Δubr1 strain expressing the misfolded substrate ΔssCL*myc, Δubr1 cells containing the luciferase-based substrates show increased growth on medium lacking leucine indicating an involvement of Ubr1 in the degradation process. Using yeast strains lacking the Hsp31 chaperone family only small effects can be detected when expressing the substrate LucLeu2myc. Also the strain lacking both Ubr1 and the Hsp31 chaperones shows similar growth compared to the Δhsp31-33 strain. The effect of Ubr1 was stronger when the steady state level of the mutated version of luciferase, LucDMLeu2myc, is monitored. Also the influence of the Hsp31 chaperone family on the steady state level of LucDMLeu2myc is enhanced as indicated by a clear increase of growth of the Δhsp31-33 deletion strain compared to wild type. Overexpression of the substrates causes similar growth of all used yeast strains (Fig. 3.41 lower panel). Due to the high substrate protein concentration even in the wild type strain a high level of growth can be observed on plates lacking leucine. These results fit to the cycloheximide chase experiments where no differences in substrate degradation among the used strains can be observed when the substrates are overexpressed (Fig. 3.11).

The same strains expressing both the substrates LucLeu2myc or LucDMLeu2myc were used for luciferase activity assays. The assays were performed using cell lysates obtained either from exponential growing cells or stationary cells in glucose-containing medium.

Figure 3.42: The cell lysate of a LucDMLeu2myc-expressing strain absent in both Ubr1 and the Hsp31 chaperones exhibits a dramatic increase of luciferase activity when grown to stationary phase. 10 OD600 of cells were harvested either in exponential phase at an OD600 of approximately 1.0 or after 72 h of growth at 30

°C prior to cell lysis and luciferase activity measurements. The relative light units (RLU) were normalized to the total protein concentration of each cell lysate by dividing by the protein concentration of each cell lysate. Plotted data represent the mean of three luciferase measurements. Error bars indicate the standard error of the mean.

In Fig. 3.42 it can be observed that the specific luciferase activity of the destabilized luciferase substrate LucDMLeu2myc (LucDM) is clearly decreased in all strains compared to the relative tightly folded LucLeu2myc substrate (Luc). As expected, cell lysates obtained from strains not expressing any Luciferase substrate (pRS) only display background luciferase

activity. In exponential phase the luciferase activities differ between the used strains in a way that is difficult to interpret. Therefore, the data obtained from exponential and stationary phase were used for generating Fig. 3.43 showing the change of luciferase activity after growth from exponential to stationary growth phase. In exponential growth phase the Δubr1 strain shows the lowest luciferase activity for both substrates (Fig. 3.42 upper panel) whereas in cells grown to stationary phase a clear influence of Ubr1 on luciferase activity can be observed compared to the wild type strain. The influence of Ubr1 on luciferase activity is expected confirming previous results showing stabilization of LucLeu2myc and LucDMLeu2myc in strains lacking Ubr1 whereas degradation can be observed in corresponding wild type strains (Fig. 3.11). The stabilization of both substrates in the Δubr1 strain is also visible in Fig. 3.43 where the change of luciferase activity after 72 h of growth is displayed. A clear difference in luciferase activity between exponential and stationary phase can be observed for the quadruple deletion strain Δhsp31-33Δubr1. When expressing the destabilized substrate LucDMLeu2myc the luciferase signal in exponential phase is relatively low whereas in stationary phase cells the signal dramatically increased in the Δhsp31-33Δubr1 strain (Fig. 3.42).

Figure 3.43: Cell growth from exponential to stationary phase (72 h growth) causes a strong increase of luciferase activity in the cell lysate obtained from the Δhsp31-33Δubr1 strain expressing the mutated luciferase substrate LucDMLeu2myc. The data shown in Fig. 3.42 were used for generating this figure. The RLU/mg values obtained from stationary phase (Fig. 3.42) were divided by the corresponding values of the exponential phase. The resulting factor describes the change of luciferase activity after 72 h growth of corresponding yeast strains expressing either LucLeu2myc (upper panel) or the destabilized luciferase substrate LucDMLeu2myc (lower panel).

In Fig. 3.43 one can observe that in the wild type, Δubr1 and Δhsp31-33 strains the luciferase activity of the destabilized version LucDMLeu2myc increase only to a less extent compared to LucLeu2myc. This is probably due to increased degradation of LucDMLeu2myc compared to LucLeu2myc. This can also be observed in degradation kinetics displayed in Fig. 3.11.

Also from growth tests one may conduct that the steady state levels of LucLeu2myc in the Δubr1 and wild type strains are more similar than those of the LucDMLeu2myc substrate.

Here, growth of the Δubr1 strain expressing LucDMLeu2myc is stronger on medium lacking leucine compared to the wild type strain (Fig. 3.41). The results for the strains lacking the

Hsp31 chaperones are surprising. Whereas the ratio of increase in luciferase activity after 72 hours of growth among the wild type, Δubr1 and Δhsp31-33 strains is similar for both luciferase substrates it is not the case for the quadruple deletion strain Δhsp31-33Δubr1. For this strain expressing LucLeu2myc it can be observed that the change of luciferase activity after entry into stationary phase is in between the Δubr1 and Δhsp31-33 strain. In contrast, for the LucDMLeu2myc-expressing Δhsp31-33Δubr1 strain the luciferase activity increases more than ten fold. This indicates a great influence of the Hsp31 chaperones on the steady state level of instable or misfolded proteins when the ubiquitin ligase Ubr1 is missing.

3.5 Protein quality control of the cytoplasmic fatty acid synthase