Characterization of U. maydis Cib1 on a protein level

As opposed to other fungi in which UPR has been investigated, translation of the cib1 pre-mRNA is not prevented in U. maydis. Hence, in the absence of ER stress

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the protein Cib1^u is produced. Upon activation of IRE1, the cib1 pre-mRNA is spliced, giving rise to the intron-free cib1^s mRNA which, in turn, is translated into the bZIP transcription factor Cib1^s (Heimel et al., 2013).

Cib1^u is only produced under unstressed conditions

In higher eukaryotes, the presence of XBP1^s mostly precludes the production of XBP1^u. In case of prolonged ER stress, both proteins coexist. To determine the ratio of Cib1^u in comparison to Cib1^s under unstressed and stressed (1 µg/mL TM or 3 mM DTT for 4 h) conditions in U. maydis, the quantities of Cib1^u and Cib1^s in SG200 expressing 3xHA-cib1 controlled by the constitutive etef promoter were measured. Surprisingly, under unstressed conditions exclusively 3xHA-Cib1^u whereas under stressed conditions only 3xHA-Cib1^s was detected. This indicates that induction of ER stress efficiently promotes splicing of the cib1 pre-mRNA (Fig. 26).

Figure 26: Cib1^u is exclusively synthesized under unstressed conditions Analysis of Cib1^s and Cib1^u ratio under unstressed and stressed conditions. 3xHA-Cib1^s and 3xHA-Cib1^u were expressed in SG200 under unstressed and stressed conditions.

ER stress was induced by treatment with either TM (1 µg/mL) or DTT (3 mM) for 4 h.

Proteins were detected on a Western Blot. Ponceau staining served as a loading control.

Cib1^u is considerably less stable than Cib1^s

From S. cerevisiae and higher eukaryotes it is known that Hac1^u/XBP1^u are highly unstable. The C-terminal tail of Hac1^u in S. cerevisiae, for example, serves as a degron which results in a calculated Hac1^u half-life of 50 seconds (Di Santo et al., 2016; Yoshida et al., 2006). Such a destabilizing element has not been described for Cib1^u, yet. For this reason, first, the stability of Cib1^u in comparison to Cib1^s was analyzed in SG200 ∆cib1 strains that contain either cib1^us-3xHA or cib1^s-3xHA controlled by the arabinose inducible crg promoter. This enables the production of comparable levels of Cib1^u-3xHA and Cib1^s-3xHA. Stability of both proteins was measured under unstressed and stressed (1 µg/mL TM for 4 h) conditions. The cells were therefore treated with cycloheximide (CHX) for 15 minutes, 30 minutes, 60 minutes and 90 minutes. CHX is an antibiotic that interferes with translational elongation and is applied as an inhibitor of protein biosynthesis. Under both growth conditions, Cib1^s-3xHA was relatively stable.

A noticeable decrease of relative protein quantity was observed after about an hour. The protein degradation was accelerated by induction of ER stress.

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Figure 27: Cib1^u stability is marginally affected by ER stress

Western Blot analysis of Cib1^s-3xHA and Cib1^u-3xHA stability. (a) Unstressed or stressed (1 µg/mL TM for 4 h) SG200 ∆cib1 cells expressing either Cib1^s-3xHA or Cib1^us-3xHA were treated with CHX up to 90 minutes. Proteins were taken before the addition of CHX as well as at every indicated time point. (b) Quantification of relative protein amount in the course of CHX treatment. Ponceau staining served as a loading control for normalization. Half-life was estimated by linear interpolation.

While, the half-life of Cib1^s was above 90 minutes under unstressed conditions, it was an estimated 77 minutes under stressed conditions. Cib1^u-3xHA was much more unstable. The half-life under unstressed conditions was an estimated

14 minutes and ER stress had only a minor impact on stability (half-life of 12 minutes) (Fig. 27)

Cib1^s and Cib1^u interact via their bZIP domain

Preliminary data indicate that the formation of Cib1^u and Cib1^s heterodimers is not only mediated by the bZIP domain but also via their C-termini (Hampel, 2016).

In order to test this initial observation, co-immunoprecipitation (CoIP) analyses were performed. To this end, several U. maydis strains expressing either full-length Cib1^s (aa 1-574) or full-length Cib1^u (aa 1-434), or the N-terminus of Cib1 (aa 1-273), or the C-terminus of Cib1^s (aa 274-574) or the C-terminus of Cib1^u (aa 274-434) were generated (Fig. 28). The interaction of two proteins, one of which was tagged with 3xHA and the other one tagged with GFP, was tested in vivo (Fig. 28a). The GFP-fusion protein was always used as bait and pulled-down by GFP-Trap. As controls, strains expressing eGFP and the corresponding interaction partners to be tested were used. Interactions between Cib1^s-GFP and Cib1^s-3xHA as well as between Cib1^u-GFP and Cib1^u-3xHA were observed, strongly indicating that they are able to form homodimers (Fig. 28b). Unspecific interaction with the GFP tag alone was not observed (Fig. 28g). Furthermore, heterodimer formation between Cib1^u and Cib1^s was discovered (Fig. 28c). In addition, an interaction between Cib1^u and Cib1^s with the N-terminus of Cib1 containing the bZIP domain was identified (Fig. 28d). Unspecific interaction with the GFP tag could be excluded (Fig. 28g). By contrast, an interaction between the C-terminus of Cib1^s with Cib1^s-GFP or Cib1^u-GFP was not observed (Fig. 28e). Interestingly, a weak interaction was discovered between the C-terminus of Cib1^u and Cib1^s-GFP but not with Cib1^u-GFP (Fig. 28f). Collectively, these data imply that homo- and heterodimer formation is mainly mediated by the bZIP domain. Nevertheless, the C-terminus of Cib1^u might potentially be involved in this interaction as well.

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Figure 28: N-termini of Cib1^s and Cib1^u are crucial for homo- and heterodimer formation

Analysis of Cib1^s and Cib1^u interaction via CoIP. (a) Scheme of the CoIP procedure and the subsequent Western Blot analysis. (b) Western blot of CoIPs of Cib1^s-GFP and Cib1^s-3xHA (top) as well as Cib1^u-GFP and Cib1^u-3xHA (bottom), (c) Cib1^s-GFP and Cib1^u-3xHA as well as Cib1^u-GFP and Cib1^s-3xHA, (d) Cib1^s-GFP and Cib1-N-term-3xHA as well as Cib1^u-GFP and Cib1-N-term-3xHA, (e) Cib1^s-GFP and Cib1^s -C-term-3xHA as well as Cib1^u-GFP and Cib1^s-C-term-3xHA, (f) Cib1s-GFP and Cib1^u -C-term-3xHA as well as Cib1^u-GFP and Cib1^u-C-term-3xHA. In all cases, 1/100 of the whole protein lysate was loaded in the input lanes. IP represents the proteins bound by the GFP-Trap^® beads. Interaction was tested in three biological replicates (exception (b) top).

(g) Western Blot of control CoIPs between eGFP and Cib1 derivatives. One representative replicate for each interaction combination is shown.