GET3B interacts physically with the C-termini of ALB3 and ALB4
Due to the fact that the alb3 mutant does not survive beyond the seedling stage when germinated on soil (Sundberg et al., 1997), we did not include it in our preceding genetic analysis. Still we wanted to analyze the interplay of GET3B with ALB3 as well as ALB4 and verify if the latter two could function as the terminal stromal insertases of the pathway. We were interested in both homologues since these were suggested to engage with similar sets of interactors (Trösch et al., 2015).
To investigate the interaction between GET3B and ALB3 or ALB4, we recombinantly expressed GET3B C-terminally fused to GST (GST-Get3b) as well as C-terminal fragments of ALB3 and ALB4 N-terminally fused to a His-tag (Alb3C-His and Alb4C-His; Ackermann et al., 2021) and performed pulldown experiments (Figure 7A, B). We mixed GST-Get3b with Alb3-His as well as GST-Get3b with Alb4C-His and incubated them with Glutathione Sepharose. In order to check for unspecific interactions, Alb3C- and Alb4C-His were incubated with GST and Glutathione Sepharose resin (Figure 7A). We observed residual amounts of both Alb3C- and Alb4C-His being pulled down by Glutathione Sepharose and the GST tag, indicating a non-specific interaction with the resin. However, when GST-Get3b was included in the pulldown experiment, we observed a clear enrichment of both Alb3C- and Alb4C-His, suggesting a specific interaction between GET3B and the C-terminus of ALB3 and ALB4 (Figure 7B).
In order to corroborate the in vitro findings we employed a yeast two-hybrid split ubiquitin assay to further investigate the interaction between GET3B and ALB3 as well as ALB4. We transfected yeast cells with constructs expressing full length ALB3 or ALB4 N-terminally fused to the C-terminus of ubiquitin (Alb3-Cub and Alb4-Cub), along with GET3B fused to the N-terminus of ubiquitin (NubG-Get3b) (Figure 7C, D). As controls, we transfected cells with Alg5 fused to the N-terminus of ubiquitin, both with and without the mutation allowing spontaneous reassembly of ubiquitin (Alg5-NubG and Alg5-NubI, respectively). As additional controls, we tested the C-terminus of ubiquitin N-terminally fused to SEC1 and ALG5 (Sec1-Cub and Alg5-Cub) together with NubG-Get3b (Figure 7E).
Upon growth on the corresponding dropout medium, we observed robust cell growth in combination with NubG-Get3b and Alb3-Cub as well as Alb4-Cub fusion constructs, indicating a positive interaction (Figure 7C, D second column). In contrast, cells co-transfected with the control protein, Alg5-NubG as well as Sec1-Cub did not exhibit significant growth under the same conditions (Figure 7E second column). As positive controls, cells expressing Alg5-NubG grew on all media, confirming the functionality of the assay.
The combination of the pulldown experiments as well as the yeast two-hybrid split ubiquitin experiment indicated a physical interaction between GET3B and the C-terminus of ALB3 and ALB4. These results together with previous ones (Anderson et al., 2021) support the notion that GET3B selectively binds clients and interacts with insertases, potentially facilitating the membrane integration of proteins within chloroplasts.