Effects of fluorescent tags and activity status on the membrane localization of ROP GTPases

ABSTRACT

Plant-specific Rho-type GTPases (ROPs) are master regulators of cell polarity and development. Over the past 30 years, their localization and dynamics have been largely examined with fluorescent proteins fused at the amino terminus without investigating their impact on protein function. The moss Physcomitrium patens genome encodes four rop genes. In this study, we introduce a fluorescent tag at the endogenous amino terminus of ROP4 in wild-type and rop1,2,3 triple mutant via homologous recombination and demonstrate that the fluorescent tag severely impairs ROP4 function and inhibits its localization on the plasma membrane. This phenotype is exacerbated in mutants lacking ROP-related GTPase-activating proteins. By comparing the localization of nonfunctional and functional ROP4 fusion reporters, we provide insight into the mechanism that governs the membrane association of ROPs.

KEYWORDS:

• Cell polarity
• ROP GTPase
• moss
• fluorescent protein tagging
• membrane trafficking

Acknowledgments

Structure prediction was conducted on the bioinformatics platform of the College of Life Sciences, Sichuan University. We thank Dr. Jiang Yang and Zhiyong Tan for their kind guidance in the prediction. This project is funded by grants from the National Natural Science Foundation of China (Grant No. 32270776) and Sichuan University (Grant No. 0082604151432) to P.Y.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contribution statement

Y. conceived the project and wrote the manuscript. J.R., Z.Y., and P.Y. performed the experiments and analyzed the data.

Data availability statement

All data generated or analyzed during this study are included in this published article.

Additional information

Funding

The work was supported by the National Natural Science Foundation of China [32270776]; Sichuan University [0082604151432].