More than just a ticket canceller: The mitochondrial processing peptidase matures complex precursor proteins at internal cleavage sites
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Abstract
Most mitochondrial proteins are synthesized in the cytosol as precursors that carry N-terminal presequences. After import into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase MPP, giving rise to shorter mature proteins. Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into the mitochondrial matrix and subsequently separated into two distinct enzymes that function in arginine biogenesis. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor both at its N-terminus and at an internal site between the Arg5 and Arg6 parts. The peculiar organization and biogenesis of Arg5,6 is conserved across fungi and might preserve the mode of co-translational subunit association of the arginine biosynthesis complex of the polycistronic arginine operon in prokaryotic mitochondrial ancestors. Putative MPP cleavage sites are also present at the junctions in other mitochondrial fusion proteins from fungi, plants and animals. Our data suggest that, in addition to its role as “ticket canceller” for the removal of presequences, MPP exhibits a second, widely conserved activity as internal processing peptidase for complex mitochondrial precursor proteins.
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###Reviewer #2:
Arg5, 6, a polyprotein is cleaved to produce two proteins Arg5 and Arg6. The authors report that production of these two proteins is mediated by a mitochondrial protease that is known for its function in N-terminal cleavage.
The in vitro analysis is interesting, but the possibility of a contaminating activity cannot be ruled out. This needs to be tested by additional experiments, preferably by more data in intact cells.
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###Reviewer #1:
This study investigates the biogenesis of Arg5,6 in the yeast S. cerevisiae. Arg5,6 is a polyprotein that was previously established to be proteolytically processed into two proteins (Arg5 and Arg6) that are part of a complex with Arg2. The primary advance reported in the current study is to assign this processing to MPP, a mitochondrial protease known primarily for removing the N-terminal signal peptides from mitochondrial precursors. Additional work showed that the cleavage occurs at an internal sequence that resembles a mitochondrial targeting sequence (MTS), which presumably explains why it is recognised by MPP. This MTS-like internal processing signal is ineffective for directing translocation on its own. Some species contain this polyprotein organisation of Arg5,6, whereas other species encode the two proteins as …
###Reviewer #1:
This study investigates the biogenesis of Arg5,6 in the yeast S. cerevisiae. Arg5,6 is a polyprotein that was previously established to be proteolytically processed into two proteins (Arg5 and Arg6) that are part of a complex with Arg2. The primary advance reported in the current study is to assign this processing to MPP, a mitochondrial protease known primarily for removing the N-terminal signal peptides from mitochondrial precursors. Additional work showed that the cleavage occurs at an internal sequence that resembles a mitochondrial targeting sequence (MTS), which presumably explains why it is recognised by MPP. This MTS-like internal processing signal is ineffective for directing translocation on its own. Some species contain this polyprotein organisation of Arg5,6, whereas other species encode the two proteins as separate open reading frames. S. cerevisiae Arg5,6 can be replaced effectively by two separately encoded products.
Specific points:
The authors use purified MPP to show that in vitro synthesized Arg5,6 precursor can be processed to the correct sized products. At that point, the authors "conclude that Arg5,6 is imported into the mitochondrial matrix and processed twice by MPP". This is plausible, but is premature based on the data, which show that MPP is able to process Arg5,6. However, the conclusion that MPP actually does process Arg5,6 in vivo is not documented, and the alternative that something else does this job is not formally excluded. This caveat should be acknowledged unless the authors are able to show necessity of MPP, not just sufficiency.
The experiment showing cleavage with purified MPP (Fig. 1E and S1A) would be strengthened with control experiments using a catalytically inactive mutant of MPP, and a Arg5,6 substrate with a mutated site for cleavage. The first control would rigorously exclude any contaminants, and the second would help verify the site of cleavage.
The conclusion that MPP processes Arg5,6 at the correct site in their in vitro experiments is based on size by SDS-PAGE. The resolution is not sufficient to draw this conclusion, which should be adjusted to say that processing occurs at approximately the correct site (unless the authors perform additional analysis to document the precise cleavage site). Mutagenesis of the putative site (point 2 above) would also be helpful in establishing the site more precisely.
The smaller products seen in Fig. 1E would seem to suggest that MPP exhibits a degree of promiscuity in vitro that is not seen in vivo. This should be noted in the text.
The authors observe that Arg6(1-343) cannot replace Arg6(1-502). They conclude that residues 344-502 are needed for enzyme activity, but this could be for many reasons. For example, Arg6(1-343) might not associate with Arg5. It is premature to imply that catalytic activity is impaired without making such measurements. The conclusion should be adjusted.
It is worth testing whether Arg5(344-862) produced by in vitro translation can be processed by purified MPP. This would help distinguish between some intrinsic problem with access versus a more nuanced issue relating to how import is mediated by the iMTS-L versus a bona fide MTS (e.g., with only the latter recruiting MPP as speculated by the authors).
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
There were some technical concerns regarding the confidence with which the authors draw conclusions about whether the MPP is indeed the responsible protease. It is likely that the authors will be able to address these concerns with relatively straightforward additional experiments.
We feel that the notion of a polyprotein being processed into multiple functional products by cellular proteases is very well established. Providing an additional example relevant for some species, but not others, is a modest …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
There were some technical concerns regarding the confidence with which the authors draw conclusions about whether the MPP is indeed the responsible protease. It is likely that the authors will be able to address these concerns with relatively straightforward additional experiments.
We feel that the notion of a polyprotein being processed into multiple functional products by cellular proteases is very well established. Providing an additional example relevant for some species, but not others, is a modest advance in our opinion, as emerged during discussion among the referees and editors. This problem is further compounded by a very similar concept for another mitochondrial protein reported by a subset of these authors recently.
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