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The dynamic movement of proteins within and between cellular compartments is essential for various biological processes, including signaling, regulation, and homeostasis. While long-distance transport often relies on molecular motors and the cytoskeleton, the mechanisms underlying short-distance vesicle transport within subcellular compartments remain poorly understood. Recent advances highlight the importance of protein phase separation and introduce the cutting-edge technology of TransitID for mapping protein trafficking dynamics.

The Role of Protein Phase Separation

Protein phase separation refers to the spontaneous formation of liquid droplets from a mixture of proteins and other biomolecules. This phenomenon has been increasingly recognized in the organization and function of membraneless organelles, such as stress granules and nuclear bodies.

A study by Qiu et al. suggests that protein phase separation also plays a pivotal role in short-distance vesicle transport within subcellular compartments. They found that synaptic vesicles (SVs) undergo rapid movements between different presynaptic compartments, such as the reserve pool and the readily releasable pool, in response to calcium signaling. Piccolo (Pclo), a giant scaffold protein, has been identified as a key player in this process. Pclo forms a phase-separated complex with SVs and other synaptic proteins, and calcium signaling triggers the extraction of SVs from the reserve pool condensate and their deposition onto the surface of the active zone condensate. This dynamic redistribution of SVs is crucial for synaptic transmission and plasticity.

In addition, the Trk-fused gene (TFG) has been implicated in the sorting and transport of COPII vesicles from the endoplasmic reticulum (ER) to the Golgi apparatus. TFG forms phase-separated droplets that coacervate with COPII vesicles, facilitating their transport in a cytoskeleton-independent manner. These examples demonstrate that protein phase separation can effectively mediate short-distance vesicle transport by organizing vesicles into distinct condensates and facilitating their interaction with specific target compartments.

Introduction of TransitID Technology

Understanding the mechanisms of short-distance vesicle transport requires sophisticated approaches to visualize and quantify protein movements within and between cellular compartments. While protein phase separation provides a compelling explanation for the organization and function of membraneless organelles, direct evidence for its involvement in vesicle transport is limited. Here, we introduce a cutting-edge technology that enables the mapping of protein trafficking dynamics in living cells with unprecedented precision.

A groundbreaking work by Qin et al. introduced a novel technology called TransitID, which enables the unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. It utilizes tandem proximity labeling with orthogonal enzymes, such as TurboID and APEX2, to identify proteins that move between specific compartments or cells over a user-defined chase period. This approach provides valuable insights into the dynamic movement of proteins and their functional implications in various cellular processes.

Applications of TransitID

Mitochondrial Protein Trafficking: TransitID has been used to map the spatial origins of nuclear-encoded mitochondrial proteins. It revealed that a subset of these proteins is translated locally near the outer mitochondrial membrane (OMM), facilitating their co-translational import into the mitochondrion. This local translation mechanism might be crucial for the efficient assembly and function of mitochondrial proteins.

Stress Granules (SGs): Stress granules are membraneless organelles that sequester mRNA and ribosomes in response to cellular stress. TransitID has been employed to map the proteome dynamics between SGs and other compartments, such as the nucleolus and the nucleus. It revealed a significant relocalization of various proteins, including transcription factors and RNA-binding proteins, to SGs during stress. This relocalization might protect these proteins from aggregation and degradation and facilitate their role in stress adaptation.

Intercellular Protein Communication: TransitID has also been used to study intercellular protein communication, particularly in the tumor microenvironment. It identified proteins transferred between tumor cells and macrophages, such as NEDD8 and PRDX1, which might play roles in immune evasion and tumor development. Additionally, it captured cytokines secreted by macrophages and their binding to receptors on tumor cells, highlighting the complexity of intercellular signaling in cancer.

Conclusion

Protein phase separation and the emerging technology of TransitID offer valuable tools for understanding the complex mechanisms of short-distance vesicle transport within cellular compartments. These advancements shed light on the role of protein condensates in organizing and directing vesicle movements and provide insights into the dynamic nature of protein trafficking in various cellular processes. Further research is needed to explore the molecular mechanisms underlying protein phase separation-mediated vesicle transport and to exploit TransitID for investigating protein trafficking dynamics in diverse biological contexts.

Reference:

1. Qiu H, Wu X, Ma X, Li S, Cai Q, Ganzella M, Ge L, Zhang H, Zhang M. Short-distance vesicle transport via phase separation. Cell. 2024 Apr 25;187(9):2175-2193.e21.

2. Qin W, Cheah JS, Xu C, Messing J, Freibaum BD, Boeynaems S, Taylor JP, Udeshi ND, Carr SA, Ting AY. Dynamic mapping of proteome trafficking within and between living cells by TransitID. Cell. 2023 Jul 20;186(15):3307-3324.e30.