N-terminal Tail Export from the Mitochondrial Matrix

Abstract

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N-and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for secindependent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.

The Thylakoid ΔpH-dependent Pathway Machinery Facilitates RR-independent N-Tail Protein Integration

“…The fact that mPftf must translocate a large N-tail to the lumen suggested that the chloroplast Oxa1p homologue might be involved. In mitochondria, Oxa1p has been shown to facilitate N-tail translocation of moderately sized amino-flanking peptides across the inner membrane (5,36,37), and antibody inhibition studies show that the chloroplast homologue is involved in integration of the membrane protein LHCP into thylakoids (38). However, recent experiments in our lab did not find an effect of antibodies to cpOxa1p on mPftf integration.…”

Protein Export across the Inner Membrane of Mitochondria

“…About 60% of this protein was integrated into the membrane in wild type mitochondria which was about twice as in oxa1 ts mitochondria. It was reported before that replacement of one negative charge by a positive charge (E77K mutant) results in significant lower insertion rates of the protein (28). Interestingly, this mutant form, however, completely bypassed the need for Oxa1 function, suggesting that the export of a positive charged protein domain can occur independently of Oxa1.…”

“…It was shown before that the charge of translocated protein domains plays a crucial role in the export process (27); the export reaction becomes more efficient with increasing negative net charge of translocated protein domains (28). Both the N and the C termini of Cox2 are of highly acidic nature and contain a large number of negative charges.…”

“…For this we employed mutants of the nuclear encoded Oxa1 substrate subunit 9 (2) because the manipulation of mitochondrial DNA is difficult and, especially in the case of Cox2, also affects protein synthesis (14). The precursor protein pSu9-(1-112)-DHFR and mutants thereof (28) were synthesized in reticulocyte lysate in the presence of [ 35 S]methionine (Fig. 5A).…”

“…In the E77K mutant, the N terminus has a net charge of ϩ1, and in the K80N mutant it has a net charge of Ϫ2. Besides the indicated mutations, both proteins are identical to Su9-(1-112)-DHFR (28). B, wild type (wt) and oxa1 ts mitochondria were preincubated at 37°C for 10 min.…”