Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

Abstract

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

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

“…Unlike proteins targeted to the interior compartments of the chloroplast, Toc75 uses an additional targeting peptide directly following the transit peptide. This peptide contains a polyglycine stretch that is required for sorting to the envelope (Inoue and Keegstra, 2003;Endow et al, 2016). It is unclear how much of the mature region of Toc75 passes the OM before insertion.…”

Plastid fatty acid export (FAX) proteins inArabidopsis thaliana- the role of FAX1 and FAX3 in growth and development

“…In consequence, this topology and the homo-oligomerization of Mic10 are prerequisites for membrane curvature in the cristae junctions of inner mitochondrial membranes Meinecke, 2016, Könnel et al, 2019). Further, the two predicted IE transmembrane domains of the plastid IMS-localized TGD5 protein also contain glycine-rich repeats (Fan et al, 2015). Here Fan and co-workers speculate that these motifs contribute to TGD5 homo-oligomerization and thereby to assembly of large TGD protein complexes by bridging the TGD1-3 ABC transporter at the plastid IE with the TGD4 ß-barrel at the OE for import of eukaryotic lipids into chloroplasts (LaBrant et al, 2018).…”

Chaos, Order and Systematics in Evolution of the Genetic Code

“…We posit selections for pre-biotic polyglycine that subsequently drove evolution of the genetic code. First, polyglycine could function as a hydrogel in forming LLPS droplets [28,[116][117][118]. Also, polyglycine can form amyloid accretions that could act in a protocell as modified hydrogels [119].…”