Uncovering the Roles and Regulatory Mechanism of the TOR-Ypk1-Fpk1 Signaling Pathway in Controlling Flippase Complexes

Uncovering the Roles and Regulatory Mechanism of the TOR-Ypk1-Fpk1 Signaling Pathway 

in Controlling Flippase Complexes

Yujin Lee1, Seung-Heon Lee1, Minjae Lee1, Eun Jung Thak2, Kyung-Tae Lee3, J. Andrew Alspaugh4,5, Yong-Sun Bahn1*

1Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

2Department of Life Science, Chung-Ang University, Seoul, South Korea

3Korea Zoonosis Research Institute, Jeonbuk National University

4Department of Medicine, Duke University School of Medicine, Durham, North Carolina, United States of America 

5Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America

*Email: ysbahn@yonsei.ac.kr

Crypococcus neoformans is an opportunistic fungus, primarily causing meningitis in immunocompromised individuals. A crucial mechanism for cellular homeostasis and signaling in this fungus involves the transport of phosphatidylserine and phosphatidylethanolamine by flippase from the plasma membrane’s outer leaflet to the inner leaflet. Our study investigates the regulatory function of the TOR-Ypk1-Fpk1 signaling pathway on flippase activity in C. neoformans. The antibiotic duramycin, a phosphatidylserine binder, served as our test agent. Our results indicate that Ypk1 acts as a positive regulator for Fpk1, thus modulating flippase activity, as evidenced by the restoration of duramycin resistance through FPK1 overexpression in ypk1Δ. This trend was also observed when exposed to osmotic stressors like NaCl and membrane stressors like SDS. Phagocytic killing tests showed an increased vulnerability in ypk1Δ compared to the wild type, while FPK1 overexpression in ypk1Δ negated this susceptibility. However, the similarity between the phagocytic killing of fpk1Δ and the wild type suggests additional regulatory factors for the flippase pathway beyond Fpk1. Our research also indicates the functional role of Ypk101, a paralog of Ypk1, in restoring partial growth under osmotic stress (1 M NaCl and KCl) conditions when overexpressed in ypk1Δ. Furthermore, pdk1Δ showed higher duramycin sensitivity than the wild-type, but YPK1 overexpression was able to restore resistance, revealing Pdk1 as a positive regulator of Ypk1 and a modulator of flippase activity. The aim of this study is to explore the TOR-Ypk1-Fpk1 pathway, presumed to regulate the flippase complex in C. neoformans, and to elucidate the interactions between these components.