Rho signaling participates in membrane fluidity homeostasis.

Daniel Lockshon,Brian K Kennedy,Alexey J Merz,Carissa Perez Olsen,Daniel A Lorenz,Andrei Chertov,Christopher L Brett,Marc R Van Gilst,Robert Alan Arkowitz

doi:10.1371/journal.pone.0045049

Abstract

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast's sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.

Highlights

Lipid bilayers must remain impermeable to even the smallest ions, yet must maintain sufficient disorder to preserve the fluidity required for dynamic processes such as migration of proteins within the membrane
This study has used genetic methods to identify a set of yeast signaling proteins that prevent C16:1 and C18:1, the main unsaturated fatty acids in yeast phospholipid, from differentially influencing growth
All but one of the signaling proteins identified in the screen are components of the cell wall integrity (CWI) pathway, the yeast Rho1-dependent MAP kinase cascade previously thought to be responsible exclusively for maintaining the integrity of the cell wall

Summary

Introduction

Lipid bilayers must remain impermeable to even the smallest ions, yet must maintain sufficient disorder to preserve the fluidity required for dynamic processes such as migration of proteins within the membrane. Such homeostasis is critical for proper receptor signaling, membrane curvature, endocytosis, exocytosis, and organelle biogenesis. The increase in width of the B. subtilis cell membrane that accompanies loss of fluidity induces autophosphorylation of DesK, a histidine kinase sensor [2], and the ensuing phosphorylation of the transcriptional activator DesR elicits transcription of des, the sole acyl desaturase. The resulting increase in monounsaturated relative to saturated fatty acids within B. subtilis phospholipid disrupts acyl chain packing to restore fluidity. While the compensatory changes in phospholipid acyl composition that occur in response to alterations in temperature (often termed homeoviscous adaptation [3]) are well established [4,5,6], the signaling pathways that achieve such homeostasis have not been identified

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Results

Conclusion