Abstract
The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.
Highlights
The light-harvesting chlorophyll a/b protein (LHCP) organelle, proteolytically processed to remove the aminois synthesized in the cytosol as a precursor terminal transit peptide, and fully assembled into thylakoids that is imported into chloroplasts and assembled into (4)
All assays received 4000molecules pLHCP/chloroplast equivalent and approximately 35 mM K+ ions derived from the assay and resuspension buffer
The quantity of pLHCP is displayed by the bars with fill, of mature size LHCP by bars with EZl fill, and of LHCP-DP, the characteristic proteasedigestion product, by bars with R fill
Summary
The light-harvesting chlorophyll a/b protein (LHCP) organelle, proteolytically processed to remove the aminois synthesized in the cytosol as a precursor (pLHCP) terminal transit peptide, and fully assembled into thylakoids that is imported into chloroplasts and assembled into (4). Integration assays were conducted by incubating chloroplast lysates (100 pg of chlorophyll) with pLHCP and 10 mM Mg-ATP in 300 pl of import buffer plus 10 mM MgClz for 30 min at 25 “C.Thylakoids were recovered by centrifugation and either washed with import buffer, extracted with 0.1 M NaOH, or treated with thermolysin as described (4).
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