A Ycf2-FtsHi Heteromeric AAA-ATPase Complex Is Required for Chloroplast Protein Import

Abstract

Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.

Major Changes in Plastid Protein Import and the Origin of the Chloroplastida

“…Mutations in one of the two copies led to an independence of F-based targeting, alternative substrate recognition, the emergence of NTS phosphorylation (Garg and Gould, 2016), and a cytosolic 14-3-3/Hsp70-based guidance complex (May and Soll, 2000) that we predict is unique to the green lineage, too. The plastid-encoded Tic214(YCF1)/YCF2/FtsHi complex emerged early in chlorophyte evolution, too, maybe through the duplication of an early Tic20-like protein (Wunder et al, 2007;Kikuchi et al, 2018). The components of this complex are highly diverse, except for a C-terminal motif, and were entirely lost in grasses without impacting protein import (de Vries et al, 2015(de Vries et al, , 2017.…”

Major changes in plastid protein import and the origin of the Chloroplastida

“…Mutations in one of the two copies led to an independence of F-based targeting, alternative substrate recognition, the emergence of NTS phosphorylation 33 , and a cytosolic 14-3-3/Hsp70-based guidance complex 105 that we predict is unique to the green lineage, too. The plastid-encoded Tic214(YCF1)/YCF2/FtsHi complex emerged early in chlorophyte evolution, too, maybe through the duplication of an early Tic20-like protein 26,106 . The components of this complex are highly diverse, except for a C-terminal motif, and were entirely lost in grasses without impacting protein import 107,108 .…”

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

“…Tic32, on the other hand, is a non-essential regulatory subunit with a versatile enzymatic activity (Balsera et al 2010) that could readily serve some TIC-independent purpose. The recently characterized components of the plastid import machinery conserved in plants and green algae, namely Tic236 (Chen et al 2018) and a heteromeric AAA-ATPase complex consisting of Ycf2 and FtsH-like proteins (Kikuchi et al 2018), are likewise not discernible in the proteome or transcriptome (Záhonová et al, 2018), providing additional support for the notion that the plastid protein import mechanism in euglenophytes differs profoundly from that of other eukaryotes.…”