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Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.

The ubiquitin proteasome system (UPS) is a complex cellular machinery that catalyzes degradation of misfolded or damaged proteins and regulates turnover of native proteins in eukaryotic cells, thus playing a crucial role in maintaining protein homeostasis. The UPS has emerged as a drug target for a diverse range of diseases characterized by accumulation of misfolded or aggregated proteins. While enhancement of UPS activity is widely recognized as a promising strategy to prevent accumulation of aberrant, off-pathway protein conformations and ameliorate the phenotypes of a wide range of protein misfolding diseases, the molecular mechanisms underlying activation of proteasomal degradation are poorly characterized. We report the development of a yeast selection platform for genome-wide selection of UPS activators. We engineered the Saccharomyces cerevisiae selection marker orotidine-5'-phosphate decarboxylase (URA3) to function as a substrate of proteasomal degradation through fusion to UPS-sensitive tags. The resulting UPS-sensitive URA3 variant links UPS activity to cell growth. The yeast selection platform reported in this study will open the way to high-throughput, genome-wide studies aimed at identifying modulators of UPS function that might provide novel target for therapeutic applications.