Dbp2 is a DEAD-box RNA helicase in S. cerevisiae, whose main function is connected to the ribosomal RNA processing and transcription. Interestingly, the localization of Dbp2 is also highly dependent on the presence of glucose, which enables us to study how cellular stress impacts gene expression machinery. Studies from the Tran laboratory show that the rapid nuclear export of Dbp2 in the absence of glucose. Furthermore, unpublished studies from the Tran lab have linked specific phosphorylation events on Dbp2 to glucose-dependent localization. To determine if phosphorylation affects Dbp2’s function and localization, our first step is to generate mutant Dbp2 proteins that localize to the nucleus or the cytoplasm, irrespective of the presence of glucose. To this end, site-directed mutagenesis was used on plasmids containing wild type DBP2 genes to convert codons encoding select serine residues to codons that would encode amino acids mimicking the dephosphorylated and phosphorylated state of Dbp2. Following transformation of these mutant-expressing plasmids and a wild type DBP2 as a control into a dbp2∆ strain, a serial dilution spot assay was performed on glucose and galactose plates (mimicking – glucose). We observed that both the phosphorylation mutants (both the non-phosphorylatable and the phosphor-mimetic) grew substantially better than our wild type Dbp2 and empty vector. This was unexpected and will be followed up with future studies in the Tran laboratory. In addition to mutant Dbp2 generation, we also set out to identify protein interaction partners of Dbp2 in the cytoplasm. Our goal was to use proximity-dependent biotin identification (BioID) using an AirID biotin ligase tag to identify Dbp2’s cytoplasmic interaction partners. Following unsuccessful isolation of proteins, immunofluorescence was performed to verify the localization of the ligase tag. Immunofluorescence indicates the AirID tag is more cytoplasmically localized in comparison to nuclear Dbp2, thus, the study was inconclusive. Due to the mislocalization of the AirID tag, in future studies we plan to use different versions of the biotin ligase tag such as the TurboID tag.
RL was supported by an NSF REU site award (DBI 1757748)
