Structural Analysis of Tha4, a Twin-arginine Translocase Protein Localized in Plant Thylakoid Membranes

Abstract

The chloroplast has a complex structure with multiple internal thylakoid membranes surrounding internal lumenal compartments. Chloroplast proteins are localized to these suborganellar locations via different protein targeting mechanisms. In this study, we investigated the three-dimensional (3D) structure of Tha4, an essential component of the twinarginine translocation (Tat) system of thylakoid membranes that mediates protein targeting to the lumen. Full-length Tha4 fused with green fluorescent protein (GFP) localized to thylakoid membranes with a discrete punctate staining pattern when expressed transiently in protoplasts. The transit peptidedeleted mature form of Tha4 was expressed in Escherichia coli, yielding multiple high molecular weight complexes in vitro. These complexes adopt ring-shaped structures of varying sizes, and long filamentous structures were also evident. Electron microscopy and image processing analyses revealed a roughly triangular ring-shaped structure, with one end of the complex open, and the other closed, based on the electron density map. The height of the cylindrical pore is 47 angstrom, comparable to the thickness of a typical lipid bilayer.