Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions

Abstract

The precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and a modified pSS containing a C-terminal hexahistidyl tail (pSS(His) 6 ) were imported into isolated Chlamydomonas chloroplasts with comparable efficiency. In the presence of Ni 2+ ions the import of pSS(His) 6 was inhibited and the precursor bound to the envelope remained protease sensitive, while import of pSS was not affected. Addition of an excess of i -histidine suppressed the inhibition demonstrating that the hexahistidyl-Ni 2+ complex was responsible for import inhibition. Inhibition could be observed between about 0.5 and 10 mM Ni 2+ , depending on the total protein content in the assay. Import incompetent Ni 2+ -precursor complexes can be used to study early events in chloroplast protein import.© 1997 Federation of European Biochemical Societies.

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas

“…The linearized plasmid pSSpSP64 containing the rbc S2‐gene (kind gift of M. L. Mishkind, Rutgers University, NJ, USA) was transcribed in vitro and translated in a wheat germ system in the presence of [ 35 S]methionine [30]. For calculation of the binding parameters of pSS to chloroplasts the absolute amount of pSS synthesized in the wheat germ system had to be determined [9].…”

Targeting of proteins into and within the chloroplast

“…On the other hand binding of specific antibodies to the DHFR moiety or to the protein A moiety of a precursor-protein A fusion protein arrested import (Schnell andBlobel 1993, Wu et al 1996). Also consistent with an unfolding/refolding model is the inhibition by glyphosphate of the import of the precursor for 5-enolpyruvylshikimate-3-phosphate synthase (della Cioppa and Kishore 1988) or the inhibition by Ni 2+ ions of the import of the small subunit of ribulose-1,5-bisphosphate carboxylase containing a C-terminal hexahistidyl tail (His)6 (Rothen et al 1996). Probably the unfolding capacity of the chloroplast envelope membrane is able to discriminate between the binding constants, thereby explaining why the import of (His)6-containing precursor protein is inhibited by Ni 2+, while DHFR fusion protein is still imported in the presence of methotrexat (Rothen et al 1996).…”

Insertion of light‐harvesting chlorophyll a/b protein into the thylakoid

“…His tags with complexed Ni 2+ ions have been used as a translocation blocker in protein import into plastids [15] and in protein transport across the cytoplasmic membrane in E. coli [16]. It is unknown how the His 6 ‐Ni 2+ complex inhibits protein translocation.…”

“…A number of such fusions were with short peptides containing six consecutive histidine residues (His tags). The His tag itself has been found to be relatively unobstructive to protein structures in a large number of recombinant proteins [15,16]. Transgenic plants carrying a gene for an N‐terminally His‐tagged Lhcb1 accumulated this protein in the thylakoid, implying that the import of this protein into the chloroplast had not been inhibited [17].…”

“…However, His tags form very stable complexes with metal ions including Ni 2+ and Cu 2+ , and these complexes are expected to be hydrophilic and rigid enough that they impair protein translocation. His tags have been shown to block the import of chloroplast precursor proteins in the presence of Ni 2+ ions [15]. The Escherichia coli outer‐membrane protein pro‐OmpA carrying a His tag in its N‐terminal translocation initiation domain is arrested in the presence of 100 µ m Ni 2+ during early events of translocation across the cytoplasmic membrane [16].…”