Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena

Abstract

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that 35S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15°C and transport to the chloroplast resumed after transfer to 26°C. Transport of the 35S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15°C cells returned to 26°C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.

Analysis of Euglena gracilis Plastid-Targeted Proteins Reveals Different Classes of Transit Sequences

“…Although the presence of a signal sequence-like region was part of our selection criteria, we found no evidence in the entire database of a plastidtargeted protein that lacked a signal sequence, suggesting that in Euglena, all plastid proteins proceed to the organelle via the endomembrane system. Although some in vitro studies have suggested the potential direct import of proteins into Euglena plastids, thereby bypassing the ER (65), the bulk of relevant biochemical work indicates that transport via the endomembrane system is required for plastid targeting (38,67,(69)(70)(71). The endomembrane system is also important for plastid targeting in all protists with complex secondary plastids, including those with three (49, 55) and four (4,7,16,19,37,59,78,79) plastid membranes.…”

“…The importance of short, cytoplasm-exposed targeting motifs for intracellular sorting is well known (9). For Euglena, Sláviková et al (67) determined that this cytoplasm-exposed portion of the presequence is not required for plastid import in vitro, but they suggested that it may function in vesicle routing.…”

“…In Euglena, the region between the signal sequence and the stop-transfer sequence in class I proteins functions as a TP (67). This region and the TPs of class II proteins possess characteristics typical of most TPs.…”

“…In both cases, plastid proteins are targeted through the endomembrane system (49,53,67,70,71). From studies of several complete, publicly available Euglena gracilis plastid protein sequences (13,25,27,28,38,44,52,56,61,64,66,73), it was predicted that the plastid proteins have an N-terminal signal sequence, an inference that was confirmed by both in vitro (38) and in vivo (70,71) experimental approaches.…”

The tiny enslaved genome of a rhizarian alga

“…Their ancestor must once have had a nucleomorph and transferred all essential chloroplast protein genes into its own nucleus, retargeted their proteins to the chloroplast, and then lost the nucleomorph entirely. It is possible that retargeting was easier for euglenoids, in which proteins have to cross only three membranes and the targeting machinery via Golgi vesicles that fuse with the outermost membrane copes with proteins temporarily stuck in vesicle membranes by hydrophobic sequences (14). It is widely assumed that chlorarachneans (20), there are three groups of photosynthetic eukaryotes.…”