Regulation of gene expression is essential for all living organisms. Transcription factors are proteins that can regulate gene expression through direct interaction with DNA and protein-protein interactions. The majority of transcription factors are localized to either the cytosol or the nucleus where they can easily access DNA. In contrast, membrane-tethered transcription factors (MTTFs) are bound to a membrane within the cell and therefore are physically restrained from entering the nucleus. In this way, MTTFs are innately dormant and must be proteolytically cleaved in order for the transcription factor domain to be released from the membrane and translocate to the nucleus where it can regulate gene expression. MTTFs have been identified across a broad range of organisms including bacteria, yeast, metazoans and plants. Though there have been 85 MTTFs predicted in Arabidopsis, only seven have been characterized to date. In a proteomic study carried out by Dunkley et al. 2006, a protein was identified as being a MYB transcription factor that was associated with the endoplasmic reticulum (ER) and was subsequently postulated to be a putative MTTF. This previously uncharacterized membrane-anchored MYB (maMYB) has two transmembrane domains and an R2R3-MYB transcription factor domain. maMYB is the only predicted membrane-anchored member of its family with homologs identified throughout the evolutionary tree including Physcomitrella patens (moss) but not in Chlamydomonas reinhardtii (algae). This pattern of evolutionary conservation suggests that maMYB may have a functional role in plant-specific processes that aided to the colonization of land by plants. The goal of this research was to characterize maMYB as an MTTF and to determine its functional role in Arabidopsis. The first aim of this project was to confirm the subcellular localization of maMYB to the ER and to determine the orientation of maMYB within the membrane. Using confocal microscopy techniques, we established that maMYB is localized to the ER with both termini, including the transcription factor domain, facing the cytosol. Once the orientation of maMYB in the membrane was determined, we wanted to establish that maMYB has transcriptional activity. By using the truncated form of maMYB (maMYB84-309), which contains the transcription factor domain but lacks the transmembrane domains (TMDs), along with confocal microscopy techniques and microarray analysis, we established that maMYB84-309 was localized to and retained in the nucleus where it regulates gene expression. The second aim of this project was to establish that maMYB-YFP gets proteolytically cleaved in vivo to allow the transcription factor domain to be released from the membrane. Using western blot analysis and an antibody specific against maMYB, truncated forms of maMYB-YFP were identified in protein lysates extracted from root tissue. Furthermore, through mutagenesis studies, it was shown that this cleavage is dependent upon the integrity of the TMD region and therefore occurs as a result of specific proteolytic activity. The third aim was to identify the functional relevance of maMYB in vivo. Through the use of knockdown lines in Arabidopsis, we found that the suppression of maMyb resulted in plants that have shorter root hairs compared with wild-type likely due to a decreased rate of root hair elongation. These findings add to the understanding of maMYB as an MTTF in Arabidopsis that likely functions in root hair elongation pathways.
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