New putative chloroplast vesicle transport components and cargo proteins revealed using a bioinformatics approach: an Arabidopsis model

Abstract

Proteins and lipids are known to be transported to targeted cytosolic compartments in vesicles. A similar system in chloroplasts is suggested to transfer lipids from the inner envelope to the thylakoids. However, little is known about both possible cargo proteins and the proteins required to build a functional vesicle transport system in chloroplasts. A few components have been suggested, but only one (CPSAR1) has a verified location in chloroplast vesicles. This protein is localized in the donor membrane (envelope) and vesicles, but not in the target membrane (thylakoids) suggesting it plays a similar role to a cytosolic homologue, Sar1, in the secretory pathway. Thus, we hypothesized that there may be more similarities, in addition to lipid transport, between the vesicle transport systems in the cytosol and chloroplast, i.e. similar vesicle transport components, possible cargo proteins and receptors. Therefore, using a bioinformatics approach we searched for putative chloroplast components in the model plant Arabidopsis thaliana, corresponding mainly to components of the cytosolic vesicle transport system that may act in coordination with previously proposed COPII chloroplast homologues. We found several additional possible components, supporting the notion of a fully functional vesicle transport system in chloroplasts. Moreover, we found motifs in thylakoid-located proteins similar to those of COPII vesicle cargo proteins, supporting the hypothesis that chloroplast vesicles may transport thylakoid proteins from the envelope to the thylakoid membrane. Several putative cargo proteins are involved in photosynthesis, thus we propose the existence of a novel thylakoid protein pathway that is important for construction and maintenance of the photosynthetic machinery.

Figure 1. Identification of putative chloroplast vesicle transport components in Arabidopsis.

Schematic work flow of the bioinformatics methods used to find putative vesicle transport proteins in chloroplasts. A, characteristic domains for cytosolic vesicle transport proteins identified using Prosite; B, Possible chloroplast vesicle components identified using yeast protein sequences as queries in PSI blast search of the Arabidopsis proteome. C, Arabidopsis cytosolic secretory pathway components used to search for putative chloroplast homologues. ≥ consensus score above or equal to 10;<consensus score below 10.

Figure 2. Identification of putative cargo proteins in Arabidopsis chloroplasts.

Schematic work flow of the bioinformatics methods used. The manual creation of the patterns was based on previous findings of motifs present in cargo proteins of the cytosolic vesicle transport system. A, transmemembrane cargo proteins’ patterns; B, soluble cargo proteins’ patterns.

Figure 3. Distribution of functions of putative cargo proteins in Arabidopsis chloroplasts.

As shown by the pie chart to the left, nearly half (45%) of the 32 putative cargo proteins are involved in photosynthesis, 19% have unknown functions, 12% are involved in transport, 12% are chaperones/proteases, 6% are involved in thylakoid biogenesis and 6% in stress responses/defense. The pie chart to the right shows that of the 45% of cargo proteins that are photosynthesis-related 16% are LHC proteins, 13% are PSII-related, 13% PSI-related and 3% components of the cytochrome b₆f complex.

Figure 4. Model for vesicle initiation and budding in Arabidopsis chloroplasts.

Nucleus-encoded transmembrane or soluble cargo proteins enter the chloroplast via the TOC/TIC machinery and by an unknown process approach cargo protein receptors (soluble cargo proteins) or are integrated into the inner envelope membrane (transmembrane cargo proteins). Vesicle initiation involves activation of CPSAR1 in its inactive state (CPSAR1-GDP) by a GEF protein similar to Sec12, causing it to attach to the inner envelope membrane in its active state (CPSAR1-GTP). The budding process involves recruitment of two coat proteins, Sec23/24 and Sec13, prior to scission.

Figure 5. Model for vesicle scission and uncoating in Arabidopsis chloroplasts.

When the two coats are in place, together with cargo receptors and possibly cargo proteins, the coat buds from the inner envelope membrane. Soon after budding the vesicle loses its coat, as CPSAR1-GTP dissociates and becomes inactive, in the CPSAR1-GDP state. The uncoated vesicle, also harboring v-SNARE and v-SNARE associated proteins for the forthcoming tethering process, moves towards the thylakoid membrane.

Figure 6. Model for tethering and docking in Arabidopsis chloroplasts.

The uncoated vesicle moves towards the thylakoid membrane, and becomes tethered to the acceptor membrane by the combined actions of Rab and tethering factors. The v- and t-SNAREs assemble into a tight bundle with the assistance of v- and t-SNARE associated proteins.

Figure 7. Model for vesicle fusion and cargo protein delivery to the thylakoid membrane in Arabidopsis.

The lipids of the vesicle fuse with the thylakoid membrane lipids and the transmembrane cargo proteins are transferred to the thylakoid membrane, whereas the soluble cargo proteins are released from the cargo receptors and delivered to the lumen.

Figure 8. Rab cycling in Arabidopsis chloroplasts.

Rab GTPase in its inactive, Rab-GDP, form is transformed into its active, Rab-GTP, form by an unidentified GEF in the stroma. A GAP promotes activity of the Rab GTPase to perform this action. To function properly the Rab GTPase needs a GDP dissociation inhibitor (GDI) displacement factor (GDF) that catalyzes dissociation of the GDI when bound to the inactive Rab-GDP.