Plastids Contain a Second Sec Translocase System with Essential Functions    

Abstract

Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane.

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase

“…This appears to be due to a breakdown of RNAi, as SCY1 null mutants are stably and uniformly chlorotic (Martin et al, 2009;Skalitzky et al, 2011). qRT-PCR analysis confirmed that both SCY1 and SCY2 transcripts had returned to wildtype levels in the green true leaves (Martin, 2010).…”

“…For example, sodium azide inhibits the thylakoid localization of the SECA1/ SCY1/SECE1-dependent plastocyanin and the 33-kD oxygen-evolving complex protein OE33 (Yuan et al, 1994) without affecting their import into the stroma. In the SEC2 system, SECA2, a stroma-localized homolog to SECA1 and Escherichia coli SECA (Skalitzky et al, 2011), could share a similar sensitivity to sodium azide.…”

“…In the case of plastid SEC2, the trans-compartment is the inter envelope space; for thylakoid SEC1, it is the thylakoid lumen. The SEC2 system is essential in Arabidopsis because null mutation of the gene for any SEC2 component results in embryo arrest at the globular stage (Skalitzky et al, 2011;Li et al, 2015). However, neither the translocase activity of SEC2 nor its substrates have been characterized.…”

“…The identified components SECA2, SCY2, and SECE2 are distantly related to thylakoid SEC1 components (SECA1, SCY1, and SECE1), the bacterial SECA, SECY, and SECE (Skalitzky et al, 2011;Li et al, 2015), and the endoplasmic reticulum Sec61a and Sec61g. SCY2 and SECE2 form a complex in the membrane (Li et al, 2015) and are localized primarily in the plastid envelope with possibly a small portion in the thylakoids (Skalitzky et al, 2011;Li et al, 2015). The SCY2/SECE2 complex is expected to serve as a protein-conducting channel similar to the orthologous components in other SEC systems.…”

“…A conserved SEC translocase system called SEC2 was described recently in Arabidopsis (Skalitzky et al, 2011;Li et al, 2015). The identified components SECA2, SCY2, and SECE2 are distantly related to thylakoid SEC1 components (SECA1, SCY1, and SECE1), the bacterial SECA, SECY, and SECE (Skalitzky et al, 2011;Li et al, 2015), and the endoplasmic reticulum Sec61a and Sec61g.…”