Over the course of the grant, OnTarget has profoundly expanded our understanding of how proteins find their way inside the cell. Through creative experimentation, systematic technology development, and the teamwork of a dedicated group of young scientists, we have:
• Revealed new targeting routes for proteins that lack conventional signals. We found that many mitochondrial proteins reach their destination without the classic “zip code” sequence previously thought to be essential. Similarly, we discovered that some peroxisomal proteins enter the organelle through unexpected, consensus-signal-independent routes and that membrane proteins can even be inserted into peroxisomes during their translation — a process previously believed to occur only on the endoplasmic reticulum (ER).
• Identified new molecular players in protein targeting, including factors such as Pex39, Mpf1, and Pex9, and decoded how these and other targeting factors selectively recognize their cargo. We also uncovered a new targeting motif for ER-surface proteins and described unique interfaces that allow import into peroxisomes without standard recognition signals.
• Clarified how cells assign “priority” when multiple proteins compete for the same import machinery. We discovered that phosphorylation of key targeting factors can shift import preferences, and even found an “anti-priority” mechanism that prevents fatal mis-delivery of proteins between the nucleus and mitochondria.
• Mapped the range of substrates handled by major protein translocons in the ER, such as Sec61, Ssh1, and EMC, defining their specificity in unprecedented detail.
• Developed powerful new technologies that now serve the entire cell biology community, including:
o A toolbox for proximity labeling and systematic identification of protein interactors.
o A bi-genomic split-GFP system that, for the first time, allows accurate mapping of mitochondrial matrix targeted proteins.
o A proteome-wide degron collection for rapid, controlled protein degradation.
o A complete yeast strain library for sensitive protein visualization and detection.
All these resources are freely distributed to the scientific community and are already used worldwide, with at least one new lab requesting them each week.
