Deciphering the principles governing robust targeting of proteins to organelles

Project Information

OnTarget

Grant agreement ID: 864068

DOI

10.3030/864068

Project closed

EC signature date 12 December 2019

Start date 1 September 2020

End date 31 August 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

WEIZMANN INSTITUTE OF SCIENCE
Israel

Periodic Reporting for period 4 - OnTarget (Deciphering the principles governing robust targeting of proteins to organelles)

Reporting period: 2025-03-01 to 2025-08-31

Summary of the context and overall objectives of the project

All living cells are defined by their organization into distinct compartments called organelles. Each organelle performs specialized functions essential for life — from chemical-energy production to waste recycling. To work properly, thousands of proteins made in the cell’s cytoplasm must be accurately delivered to their correct organelle. Mistakes in this “protein targeting” process underlie many human diseases, including cancer, diabetes, neurodegeneration, and numerous rare genetic disorders.
The OnTarget project set out to uncover how cells manage this immense logistical challenge: how newly made proteins are recognized, sorted, and sent to the right destination. Using baker’s yeast (Saccharomyces cerevisiae) as a powerful model system, our goal was to identify new targeting pathways, the molecular factors that guide them, and the rules that give certain proteins priority over others when cellular traffic becomes crowded.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The OnTarget project has not only answered long-standing questions in cell biology but has also opened entirely new research directions. The principles uncovered are broadly relevant — the same cellular logistics that keep yeast cells healthy are at work in human cells, and their failure contributes to countless diseases. By decoding these fundamental processes, our work lays the foundation for future medical and biotechnological advances, from understanding metabolic disorders to engineering cells with new capabilities.
Beyond its scientific impact, OnTarget has had an important human legacy. The project trained a generation of exceptional scientists: many of my former PhD students and postdocs have now established independent academic careers across Israel, Europe, and the United States, carrying forward the spirit of curiosity and collaboration that defined our team.

Through OnTarget, we have transformed how the field views protein targeting — from a handful of well-known pathways to a complex, dynamic network that ensures the cell’s internal organization remains robust and adaptable. We have shown that precision in cellular logistics emerges not from rigid rules but from a flexible system finely tuned by evolution.
Our discoveries, tools, and people continue to shape this vibrant field. The ERC’s long-term support has made it possible to take bold, high-risk steps — and in doing so, to bring us closer to a complete understanding of one of life’s most fundamental processes: how every protein finds its place in the cell.

Targeting of proteins to organelles is an essential yet complicated process