Yeast as a Model for SMAD Nuclear Transport
Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Khokha, Mustafa
Abstract
Transforming growth factor β (TGF-β) signaling plays an important role in tissue patterning during embryonic development. When mis-regulated, TGF- β signaling can lead to diseases such as birth defects and cancer. To transduce the ligand signal from the receptor to the nucleus, current models suggest that a complex forms between a co-SMAD (SMAD4) and receptor-regulated R-SMADs in the cytoplasm prior to nuclear enrichment and activation of target genes. However, the details of basal SMAD nuclear transport in the absence of an exogenous signal remain unclear, since the previously identified SMAD nuclear localization signal (NLS) was based on preliminary understanding of Importin-⍺/β transport and its NLSs and does not consider the current extensive repertoire of nuclear transport receptors (NTRs) and NLSs. Therefore, we sought to re-investigate the question of the nuclear transport of SMADs. For this purpose, the budding yeast Saccharomyces cerevisiae, is an ideal system to study basal SMAD nuclear localization as it removes some of the complexity inherent to vertebrate cells. Yeast have conserved nuclear transport machinery but lack TGF-β pathway members. When we introduce mammalian TGF-β pathway components into this minimal system, we find that various fluorescently tagged SMADs (eGFP-SMADs) have differential localization to the nucleus. In a Ran-dependent manner, SMAD2/3 are enriched in the nucleus while SMAD1/5 and SMAD4 are excluded. Focusing on SMAD3, an unbiased screen of all defined NTRs confirms that importin-7 and importin-8 are SMAD3 NTRs. We also show that the previously identified SMAD NLS unexpectedly localizes eGFP to the cell membrane and not the nucleus. Further domain analysis prompted us to redefine the SMAD NLS as an importin binding surface rather than as a sequence motif. Our results in yeast translate to impacts on SMAD3 function in both Xenopus and nuclear localization in mammalian cell culture. Together, these data demonstrate that our yeast model is an efficient platform to interrogate the nuclear transport of embryonic signaling effectors. By studying SMAD proteins independently from one another in a non-signaling state in this minimal system, we discovered nuances in SMAD nuclear transport that have gone overlooked in animal models.
Recommended Citation
González, Delfina P., "Yeast as a Model for SMAD Nuclear Transport" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1357.
https://elischolar.library.yale.edu/gsas_dissertations/1357
