Cell-penetrating peptides stimulate protein transport on the Twin-arginine translocation pathway: evidence for a membrane thinning and toroidal pore mechanism

Abstract

The Tat pathway is essential for photosynthetic protein transport across plant thylakoid membranes and is also ubiquitous throughout prokaryotes and archaea. The Tat pathway is unique amongst protein translocation pathways as it specializes in transporting folded proteins driven by a proton motive force. Mechanistic details of the actual translocation step (s) of the pathway remain elusive. Here, we show that membrane thinning stimulates Tat transport and, conversely, membrane strengthening abolishes Tat transport. We draw parallels from the Tat transport mechanism to that of cell penetrating peptides and propose that the Tat pore could be toroidal in shape and lined by lipids, as in those formed by cell penetrating peptides. Significance StatementProtein translocation across membranes is a significant cellular activity in both prokaryotes and eukaryotes. The Tat pathway for protein translocation operates in bacteria, archaea, chloroplasts, and plant mitochondria. Its mechanism of action has been difficult to decipher, but recent evidence suggests it does not use a conical proteinaceous transport channel. Instead, it has been suggested to translocate proteins through lipid-lined toroidal pores set up by membrane thinning. This work supports that hypothesis by showing that membrane-thinning cell-penetrating peptides stimulate the Tat pathway in both chloroplasts and bacterial plasma membranes, and that membrane stabilization blocks the pathway. We believe this is the most direct evidence to date of the toroidal pore mechanism operating in the Tat pathway.