All cellular processes are carried out by proteins, linear chains of amino acids that fold into three-33 dimensional structures. While the amino acid sequence of a protein is primarily determined by its 34 DNA sequence, many proteins are additionally modified by proteolytic cleavage after their synthesis. 35 Processing of polypeptides at their N-terminus is pervasive in both prokaryotic and eukaryotic 36 proteomes. For instance, the amino-terminal methionine is removed from many polypeptides when (Fig. 1B) . Obviously, the precursor is rapidly and 91 efficiently cleaved in vivo, which yields a C-terminal Arg5 fragment. 92 In order to analyze the mechanistic basis of this unusual biogenesis, we sought to reconstitute the 93 biogenesis and processing of Arg5,6 in an in vitro system. To this end, we synthesized radiolabeled 94 Arg5,6 precursor in reticulocyte lysate and incubated it with isolated yeast mitochondria. We 95 observed that the precursor of around 90 kDa was efficiently processed to a slightly smaller polypeptides, but not the Arg5,6 precursor were protected from digestion with externally added 100 proteinase K. This shows that these polypeptides were translocated across the mitochondrial outer 101 membrane. Both import and processing were dependent on the mitochondrial inner membrane . We conclude that Arg5,6 is imported into the mitochondrial matrix 104 via the presequence pathway and cleaved into separate polypeptides inside mitochondria.
How is the Arg5,6 precursor processed to give rise to the Arg6 and Arg5 enzymes? A number of 106 proteases in the mitochondrial matrix are described (Quiros et al., 2015 , Veling et al., 2017 . 107 However, most of them are either implicated in degradation and turnover of proteins (such as the Lon 108 protease Pim1) or known to remove short peptides or single amino acids from the N-terminus of 109 mitochondrial precursor proteins (such as Oct1 or Icp55), but not for internal cleavage of proteins and 503 (Fig. 1D ). The latter one would fit to the molecular masses of the Arg6 and Arg5 proteins 121 observed in our in vitro system. To directly test whether MPP can cleave the Arg5,6 precursor, we 122 purified MPP from E. coli expressing His-tagged Mas1 and Mas2 (the two subunits of MPP).
Incubation of radiolabeled Arg5,6 precursor protein with MPP resulted in the formation of smaller 124 fragments whose size perfectly matched those that were generated after import into isolated 125 mitochondria (Fig. 1E ). Proper processing was blocked when EDTA was added to the reaction, which 126 inhibits the metalloprotease MPP by chelating divalent cations (Suppl. Fig. 1A ) (Luciano et al., 1998) . 127 We conclude that Arg5,6 is imported into the mitochondrial matrix and processed twice by MPP. A The unusual biogenesis of Arg5,6 prompted us to ask whether Arg6 and Arg5 can also be imported 134 separately. Therefore, we created truncated versions of the ARG5,6 gene which contain only the 135 N-terminal Arg6 with its MTS (Arg6 1-502 ) or only the C-terminal Arg5, starting at the first 136 (Arg5 344-863 ) or the second iMTS-L (Arg5 503-863 ). For the latter two, we also generated variants which 137 additionally carry the well-characterized presequence of ATP synthase subunit 9 from Neurospora 138 crassa (Su9-Arg5 344-863 and Su9-Arg5 503-863 ) ( Fig. 2A) .
Radiolabeled proteins were synthesized in vitro and incubated with isolated mitochondria to test their 140 import competence. As expected, Arg6 1-502 was efficiently imported and its MTS was cleaved 141 (Fig. 2B) . The shorter Arg5 variant (Arg5 503-863 ) did not reach a protease-protected compartment and, 142 thus, was not imported into mitochondria (Fig. 2C) . However, N-terminal fusion of the Su9 143 presequence completely restored import of Arg5 503-863 (Fig. 2D) . Hence, import of Arg5 and Arg6 144 into mitochondria is in principle possible also for separated polypeptides, at least in the in vitro assay 145 used here. 146 We next tested whether Arg6 and Arg5 can be imported separately in vivo and function in arginine 147 biosynthesis. The deletion of the ARG5,6 gene renders yeast cells auxotrophic for arginine. We 148 expressed either the full length Arg5,6 precursor or combinations of separate Arg6 and Arg5 variants 149 in a Δarg5,6 deletion mutant. If Arg6 and Arg5 make their way into mitochondria and acquire a 150 functional conformation, arginine prototrophy should be restored. When we streaked out these cells 151 on plates with minimal growth medium lacking arginine, we observed growth for the wildtype and 152 the Δarg5,6 mutant complemented with full length Arg5,6, but not for Δarg5,6 carrying only an 153 empty plasmid, as expected (Fig. 2E ).
The mutant expressing both Arg6 1-502 and the shorter Arg5 503-863 variant was not able to grow without 155 arginine (Fig. 2E ). However, when the presequence of Su9 was fused to the short Arg5 503-863 , cells 156 regained arginine prototrophy (Fig. 2F ). All strains grew on plates containing arginine, showing that 157 the Arg5 503-863 protein has no toxic gain-of-function effect when residing in the cytosol (Suppl. Fig.   158 1B,C). Growing the strains in liquid medium lacking arginine confirmed the results obtained on plates 159 and additionally demonstrated that the growth rate of the strain expressing Arg6 1-502 and Su9-160 Arg5 503-863 is comparable to that of the wildtype (Suppl. Fig. 1D . Table 1A) . 205 3,666 species were identified that encode exactly one copy each of argB and argC (Suppl. Table 1F ) that were not further investigated. We 229 also predicted the intracellular localization of all proteins via TargetP. Strikingly, mitochondrial 230 localization was assigned to almost all fusion proteins of fungi, whereas most separate proteins in 231 algae were predicted to be imported into chloroplasts (Fig. 4B) .
In summary, in fungi the acetylglutamate kinase and acetylglutamyl-phosphate reductase are 233 generally encoded as a fusion protein, which is imported into mitochondria and processed twice by 234 MPP to remove its presequence and gives rise to two functional enzymes. In contrast, algae encode 235 two separate proteins which are individually imported into chloroplasts. Gamma-proteobacteria 236 express the genes from one polycistronic RNA (Fig. 4C) . profiling analysis and indeed found prominent iMTS-Ls at each of their junction sites (Fig. 5B) .
The organization as fusion protein is an elegant solution to confer mitochondrial targeting of two 257 enzymes that reside in the same compartment and even act in subsequent steps of a biochemical 258 pathway. It is still remarkable that this organization of Arg5,6 was retained during evolution even in 259 distantly related organisms, indicating that there exists a strong constraint that maintained this 260 organization for more than a billion years of evolution. To our knowledge, this is the only example 261 of a fusion of two functionally related proteins whose organization is so widely conserved across 262 eukaryote species. Eukaryotic genomes typically strongly disfavor even "milder" variants of physical 263 coupling of genes, such as an operon-like organization which is pervasively present in prokaryotes, Besides its canonical role as "ticket canceller" that clips targeting signals, the mitochondrial 277 processing peptidase MPP obviously also possesses a "tailor" activity for internal processing of 278 several precursor proteins (Fig. 5C ). This property is conserved across the fungi, plant and potentially 279 also animal kingdoms. Internal MPP cleavage requires a proximal recognition motif which appears 280 to be an iMTS-L, but remarkably also a strong N-terminal presequence. This suggests that in order to 281 access internal cleavage sites, MPP has to be loaded onto a precursor as soon as it emerges from the 282 TIM23 channel. MPP might then "scan" the still unfolded polypeptide for cleavage sites. A protein 283 with a strong presequence will efficiently recruit MPP, which enables subsequent internal cleavage 284 at an iMTS-L before this is buried by protein folding. In analyses of the N-proteome from yeast, 
Yeast strains and plasmids 298 All yeast strains used in this study were based on the WT strain BY4742 (Winston et al., 1995) . 299 Unless indicated differently, strains were grown on synthetic medium (0.17% yeast nitrogen base and 300 0.5% (NH4)2SO4) containing 2% glucose.
The Arg5,6-coding region or a fragment of it was amplified by PCR and cloned into pGEM4 The homogenate was centrifuged for 5 min at 3,500 x g at 4°C to separate cell debris and nuclei from 321 organelles. The mitochondrial fraction was isolated by centrifugation of the supernatant from the 322 previous step for 12 min at 12,000 x g at 4°C. The crude mitochondrial pellet was gently resuspended 323 in SH buffer (0.6M sorbitol, 20mM HEPES/KOH pH 7.4), centrifuged for 5 min at 4,000 x g at 4°C, 324 recovered from the supernatant by centrifugation for 12 min at 12,000 x g at 4°C and finally 325 resuspended in SH buffer. The protein concentration of the mitochondrial suspension was determined 326 by a Bradford assay and mitochondria were diluted to a final concentration of 10 mg/ml protein with 327 ice-cold SH buffer, aliquoted, frozen in liquid nitrogen and stored at -80°C. . Table 1D ).
Subject organisms that exhibited hits (at least 25% local identity and a maximum E-value of 1E-10) 379 were grouped by the number and pattern of the identified homologs, corresponding to either encoding 380 Arg5,6 as one gene or as two genes (Suppl . Table 1E exhibiting exactly one homolog to each of the two query sequences (at least 25% local identity and a 388 maximum E-value of 1E-10) were further investigated and the nucleotide distance between the 389 subject genes was calculated (Suppl . Table 1C ). Sequence pairs with a maximum distance of 30 390 nucleotides were suspected to be encoded polycistronically. All genomic distances in nucleotides 391 were derived from Refseq genome feature tables. Sequence pairs with overlapping start and end 392 position were given a distance of one nucleotide. 