The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain.

Abstract

The precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is unique; it is a polyprotein, synthesized on membrane-bound ribosomes and transported to the Golgi apparatus prior to chloroplast localization. A cDNA corresponding to the 5' end of LHCPII mRNA has been isolated and sequenced. The deduced amino acid sequence of this cDNA indicates that Euglena pLHCPII contains a 141-amino acid N-terminal extension. The N-terminal extension contains three hydrophobic domains and a potential signal peptidase cleavage site at amino acid 35. Cotranslational processing by canine microsomes removed approximately 35 amino acids from an in vitro synthesized 33-kDa pLHCPII composed of a 141-amino acid N-terminal extension and a 180-amino acid partial LH-CPU unit truncated at the beginning of the third membranespanning hydrophobic domain. Processed pLHCPII was degraded by exogenous protease, indicating that it had not been translocated to the microsomal lumen. Extraction with 0.1 M Na2CO3, pH 11.5, did not remove the processed pLHCPII from the microsomal membrane. A stop-transfer membrane anchor sequence appears to anchor the nascent protein within the membrane, preventing translocation into the lumen. Taken together, these results provide biochemical evidence for a functional cleaved signal sequence within the N-terminal extension of a Euglena cytoplasmically synthesized chloroplastlocalized protein.

A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

“…The Euglena pLHCPII is inserted cotranslationally and anchored within canine microsomal membranes, and the N-terminal presequence is removed (Kishore et al, 1993). The in vivo pulse-chase experiments demonstrated that the pSSU was targeted to the ER and Golgi apparatus before chloroplast import.…”

“…In vitro, the pLHCPII is cotranslationally inserted into canine microsomes, processed by signal peptidase, and anchored within the microsomal membrane (Kishore et al, 1993). The pLHCPII and pSSU presequences are structurally similar, containing an N-terminal signal peptide domain and an additional hydrophobic domain that could function as a stop transfer membrane anchor sequence.…”

“…Cytoplasmically synthesized chloroplast protein precursors of Euglena (Sharif et al, 1989;Chan et al, 1990;Kishore et al, 1993), dinoflagellates (Norris and Miller, 1994), and diatoms (Bhaya and Grossman, 1991;Kroth-Pancic, 1995) contain an ER-targeting signal peptide domain at the presequence N terminus. The Euglena precursor to the light-harvesting chlorophyll a/b binding protein of photosystem II (pLHCPII) (Kishore et al, 1993) and the Phaeodactylum fucoxanthin chlorophyll a/c binding protein precursor (Bhaya and Grossman, 1991) are inserted cotranslationally into canine microsomes and the signal peptide is cleaved, suggesting that in organisms whose chloroplasts are surrounded by more than two membranes, chloroplast proteins are transported cotranslationally into the ER before chloroplast localization.…”

“…(Kishore et al, 1993) presequence and include an N-terminal signal peptide domain. Taken together, the immunoelectron microscopy and in vitro import studies suggest two mechanisms for Euglena chloroplast protein import: direct import from the cytoplasm of stromal protein precursors as found in higher plants, and transport of thylakoid protein precursors to the Golgi apparatus in a membrane-bound form before chloroplast localization.…”

Bilateral Communication between Plastid and the Nucleus: Plastid Protein Import and Plastid-to-Nucleus Retrograde Signaling

“…41,42) In these organisms, plastids are also surrounded by additional membrane layers that are reminiscent of host plasma membranes. Therefore, proteins destined for these plastids are first delivered via a secretory pathway and then translocate across the typical plastid envelope, probably through the TOC-TIC pathway.…”