Evolutionary and Functional Analysis of a Chara Plasma Membrane H+-ATPase.

Suyun Zhang,Bert Van Duijn,Liam Dolan,Myckel Habets,Remko Offringa,Holger Breuninger

doi:10.3389/fpls.2019.01707

Suyun Zhang, Bert Van Duijn + Show 4 more

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https://doi.org/10.3389/fpls.2019.01707

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Abstract

H+-ATPases are the main transporters in plant and fungal plasma membranes (PMs), comparable to the Na+/K+ ATPases in animal cells. At the molecular level, most studies on the PM H+-ATPases have been focused on land plants and fungi (yeast). The research of PM H+-ATPases in green algae falls far behind due to the lack of genetic information. Here we studied a potential PM H+-ATPase (CHA1) from Chara australis, a species of green algae belonging to the division Charophyta, members of which are considered to be one of the closest ancestors of land plants. The gene encodes a 107 kDa protein with all 6 P-type ATPase-specific motifs and a long, diverse C-terminal domain. A new amino acid sequence motif R*****Q in transmembrane segment 5 was identified among the known PM H+-ATPases from Charophyta and Chlorophyta algae, which is different from the typical PM H+-ATPases in yeast or land plants. Complementation analysis in yeast showed that CHA1 could successfully reach the PM, and that proton pump activity was obtained when the last 77 up to 87 amino acids of the C-terminal domain were deleted. PM localization was confirmed in Arabidopsis protoplasts; however, deletion of more than 55 amino acids at the N-terminus or more than 98 amino acids at the C-terminus resulted in failure of CHA1 to reach the PM in yeast. These results suggest that an auto-inhibition domain is located in the C-terminal domain, and that CHA1 is likely to have a different regulation mechanism compared to the yeast and land plant PM H+-ATPases.

Highlights

Plasma membrane (PM) H+-ATPases belonging to the least divergent subfamily of the P-type ATPases, P3A, have been identified in plants, fungi, some protozoa, and archaea
PM localization was confirmed in Arabidopsis protoplasts; deletion of more than 55 amino acids at the N-terminus or more than 98 amino acids at the Cterminus resulted in failure of CHA1 to reach the PM in yeast. These results suggest that an auto-inhibition domain is located in the C-terminal domain, and that CHA1 is likely to have a different regulation mechanism compared to the yeast and land plant PM H+-ATPases
In order to identify possible PM H+-ATPase genes in C. australis, we searched the transcriptome database generated by high throughput sequencing on RNAs isolated from C. australis, using the known plant PM H+-ATPase protein sequences as bait

Summary

Introduction

Plasma membrane (PM) H+-ATPases belonging to the least divergent subfamily of the P-type ATPases, P3A, have been identified in plants, fungi, some protozoa, and archaea. The R domain in vascular plants has been shown to function as an auto-inhibition domain, containing two conserved regions (Region I and II) and a penultimate threonine (named: pT H+-ATPase) (Palmgren et al, 1991; Speth et al, 1997). This threonine can be phosphorylated by protein kinases, after which it becomes a binding site for 14-3-3 proteins (Fuglsang et al, 1999).

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