Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

Abstract

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic alpha-helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F1 beta subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.

Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts

“…[38][39][40] However, it has also been shown that some chloroplast proteins can be mistargeted to mitochondria in vitro, 10,39 whereas no mistargeting of mitochondrial proteins to chloroplasts has been observed. In order to avoid mistargeting of chloroplast proteins to mitochondria in vitro, we have developed a dual import system in which the precursor protein is incubated with a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles.…”

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the ? subunit

“…To determine how proteins are targeted to the organelles in C. reinhardtii, it is important to study directly the import of precursor proteins into C. reinhardtii organelles. We have recently initiated such studies [27] and further experiments are in progress.…”

A novelin vitrosystem for simultaneous import of precursor proteins into mitochondria and chloroplasts

“…The mitochondrial pre‐sequence of the yeast cytochrome oxidase subunit Va can function both as a mitochondrial and chloroplastic targeting peptide in transgenic tobacco (Huang et al ., 1990). The PsaF protein from C. reinhardtii was shown to be imported in vitro into spinach and C. reinhardtii mitochondria (Hugosson et al ., 1995). The transit peptides of the chloroplast outer envelope proteins, triose‐3‐phosphoglycerate phosphate translocator (TPT) and the 37 kDa protein coupled to CAT were imported into plant mitochondria in vitro but not in vivo (Brink et al ., 1994; Silva Filho et al ., 1997).…”