Targeting Determinants and Proposed Evolutionary Basis for the Sec and the Delta pH Protein Transport Systems in Chloroplast Thylakoid Membranes

Abstract

Transport of proteins to the thylakoid lumen is accomplished by two precursor-specific pathways, the Sec and the unique Delta pH transport systems. Pathway selection is specified by transient lumen-targeting domains (LTDs) on precursor proteins. Here, chimeric and mutant LTDs were used to identify elements responsible for targeting specificity. The results showed that: (a) minimal signal peptide motifs consisting of charged N, hydrophobic H, and cleavage C domains were both necessary and sufficient for pathway-specific targeting; (b) exclusive targeting to the Delta pH pathway requires a twin arginine in the N domain and an H domain that is incompatible with the Sec pathway; (c) exclusive targeting to the Sec pathway is achieved by an N domain that lacks the twin arginine, although the twin arginine was completely compatible with the Sec system. A dual-targeting signal peptide, constructed by combining Delta pH and Sec domains, was used to simultaneously compare the transport capability of both pathways when confronted with different passenger proteins. Whereas Sec passengers were efficiently transported by both pathways, Delta pH passengers were arrested in translocation on the Sec pathway. This finding suggests that the Delta pH mechanism evolved to accommodate transport of proteins incompatible with the thylakoid Sec machinery.

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

“…This view is further supported by the recent study of Henry et al (1997) in which a "dual-targeting" transit peptide was constructed that was capable of directing passenger proteins to either the sec-or ⌬pH-dependent pathways. In these experiments, however, proteins normally on the ⌬pH-dependent pathway were found to be incompatible with the sec pathway, such that dual-targeting OE17 was transported only on the former pathway.…”

A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts

“…With the exception of a synthetic peptide (see below), all reagents, enzymes, and standards were purchased commercially. Previously described plasmids were used for in vitro transcription͞translation of pLHCP (psAB80XD͞4) (13), tOE23 (14), iOE33 (15), and TM3-PC (12). However, the TM3-PC translation product, which codes for a fusion protein between the carboxyl-terminal third of pLHCP and the mature domain of plastocyanin (PCm), is designated here as pLHCP to more clearly indicate the amino acids derived from pLHCP.…”

“…Intact chloroplasts were isolated from 9-to 10-day-old pea seedlings (Laxton's Progress) and resuspended in IB. Lysates and SE were prepared from isolated chloroplasts (14,18). Chlorophyll was determined according to Arnon (19).…”

“…Assays were initiated by addition of diluted translation product (25 l) and conducted for 30 min in a 25°C water bath. Recovered thylakoids were posttreated with thermolysin, which was terminated by adding an equal volume of 50 mM EDTA in IB, collected by centrifugation, and dissociated with 50 l of SDS͞PAGE sample buffer (14). Transit complex was formed in 30-l assays by mixing 20 l of SE (0.5 mg͞ml chlorophyll equivalent) with 5 l of 30 mM Mg-ATP and 5 l of diluted translation product essentially as described (20).…”

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

“…These routes include the spontaneous insertion of proteins (18), and signal recognition particle-dependent (9), ⌬pH-dependent (6, 21), and SecA-dependent pathways (22,33). A twin-arginine motif is specifically recognized by components of the ⌬pH-dependent pathway, and hydrophobic sequences are favored by the signal recognition particle-dependent pathway (12,13). The specificity of cpSecA for the Sec-dependent chloroplast signals observed here provides a mechanism for avoiding precursor proteins with those features.…”