Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Abstract

Various chimeric precursors and deletions of the 33 kd oxygen‐evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose‐1,5‐bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N‐terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C‐terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.

Isolation and Characterization of a cDNA Clone Encoding a Member of the Com44/Cim44 Envelope Components of the Chloroplast Protein Import Apparatus

“…Radiolabeled protein precursors were prepared as reported in Wu et al (1994). Import assays were conducted as described (Ko and Cashmore, 1989). Intact chloroplasts were reisolated for further treatment and subfractionation according to Smeekens et al (1986).…”

“…Control import experiments conducted with cytoplasmic proteins such as pyruvate kinase (Wan et al, 1995) and chloroplast proteins without transit peptides (e.g. Oee1) (Ko and Cashmore, 1989), do not import nor do they bind at any level to the chloroplast. The affinity Bce44B possesses for chloroplasts is therefore genuine and did not arise from nonspecific associations.…”

Unique Translation Initiation at the Second AUG Codon Determines Mitochondrial Localization of the Phage-Type RNA Polymerases in the Moss Physcomitrella patens

“…Therefore, the majority of the proteins involved in the biogenesis of mitochondria and plastids are encoded in the nucleus, and must be translated in the cytoplasm and then imported into the respective organelles (Schatz and Dobberstein, 1996;Neupert, 1997;Soll and Tien, 1998;Keegstra and Cline, 1999). Most of these proteins have a transit peptide at the N terminus that is necessary for their import into the target organelles (von Heijne, 1986;van Loon et al, 1988;Hand et al, 1989;Ko and Cashmore, 1989;Sidorov et al, 1999). The localization of these organellar proteins has been analyzed by in vitro import experiments using isolated organelles and in vivo experiments using a reporter, such as green fluorescent protein (GFP).…”

The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems.

“…The transit signals, which are hydrophilic, basic and enriched in hydroxylated residues, are structurally and functionally equivalent to the presequences of imported stromal proteins (Ko and Cashmore, 1989;von Heijne et al, 1989;Hageman et al, 1990). In contrast, thylakoid transfer signals are strikingly similar in structural terms to signal sequences which direct the translocation of proteins across the endoplasmic reticulum and the bacterial plasma membrane (von Heijne et al, 1989).…”