Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus

Abstract

Chloroplasts evolved from a cyanobacterial endosymbiont that resided within a eukaryotic cell. Due to their prokaryotic heritage, chloroplast outer membranes contain transmembrane b-barrel proteins. While most chloroplast proteins use N-terminal transit peptides to enter the chloroplasts through the translocons at the outer and inner chloroplast envelope membranes (TOC/TIC), only one b-barrel protein, Toc75, has been shown to use this pathway. The route other b-barrel proteins use has remained unresolved. Here we use in vitro pea (Pisum sativum) chloroplast import assays and transient expression in Nicotiana benthamiana to address this. We show that a paralog of Toc75, outer envelope protein 80 kD (OEP80), also uses a transit peptide but has a distinct envelope sorting signal. Our results additionally indicate that b-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.

Toc75‐V/OEP80 is processed during translocation into chloroplasts, and the membrane‐embedded form exposes its POTRA domain to the intermembrane space

“…2), likely after import. This observation is consistent with a recently published observation [27]. There are two likely candidates for this process: (a) the stromal processing peptidase in case the N-terminal region is transferred into the stroma [27]; or (b) the plastidic type 1 signal peptidase previously identified to be involved in maturation of atToc75-III [24].…”

“…To provide independent evidence in this discussion, we reinvestigated the presence of a cleavable N-terminal portion and the topology of Toc75-V. We confirm the suggested existence of a cleavable N terminus by in vivo import experiments and by N-terminal sequencing of the protein in endogenous membranes. Moreover, we confirm that the soluble POTRA domains of Toc75-V are oriented toward the intermembrane space, which strengthens the current topology model derived by protease protection [11] and import experiments [27]. The implications for possible structural models are discussed.…”

“…There are two likely candidates for this process: (a) the stromal processing peptidase in case the N‐terminal region is transferred into the stroma ; or (b) the plastidic type 1 signal peptidase previously identified to be involved in maturation of atToc75‐III . The recently published observation suggests that the cleavage is catalyzed by the stromal processing peptidase .…”

“…The signal is cleaved by the stromal and the intermembrane space localized type I signal peptidase . In contrast, Toc75‐V does not contain such a bipartite signal and even the presence of a cleavable N‐terminal transit peptide is under debate . Initially, it was concluded that Toc75‐V in A. thaliana does not contain a cleavable signal.…”

“…Again, the same authors challenged the in vitro import results as well and suggested the presence of a cleavable signal . Finally, a recent approach by these authors using in vitro import and stromal processing assay provided further support for the existence of a cleavable signal . Before, mutant versions of Toc75‐V were generated utilizing the second ATG as start codon.…”