Redundant Mitochondrial Targeting Signals in Yeast Adenylate Kinase

Schricker, Roland; Angermayr, Michaela; Strobel, Gertrud; Klinke, Sigrid; Korber, Dorothee; Bandlow, Wolfhard

doi:10.1074/jbc.m201561200

Cited by 18 publications

(19 citation statements)

References 59 publications

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“…In the second scenario, some precursor Nfs1 molecules never reach the mitochondria and are directly targeted from the cytosol to the nucleus (2,5). A somewhat similar mechanism has been described for the protein Adk1 in its distribution between the cytosol and mitochondria (29). The nuclear fraction according to this mechanism should contain the full-length Nfs1 precursor, including the MTS.…”

Section: Nfs1 Is Processed By Two Peptidases Mpp and The Novel Icp55-mentioning

confidence: 99%

Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

Naamati

Regev‐Rudzki

Galperin

et al. 2009

Journal of Biological Chemistry

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In eukaryotes, each subcellular compartment harbors a specific group of proteins that must accomplish specific tasks. Nfs1 is a highly conserved mitochondrial cysteine desulfurase that participates in iron-sulfur cluster assembly as a sulfur donor. Previous genetic studies, in Saccharomyces cerevisiae, have suggested that this protein distributes between the mitochondria and the nucleus with biochemically undetectable amounts in the nucleus (termed "eclipsed distribution"). Here, we provide direct evidence for Nfs1 nuclear localization (in addition to mitochondria) using both ␣-complementation and subcellular fractionation. We also demonstrate that mitochondrial and nuclear Nfs1 are derived from a single translation product. Our data suggest that the Nfs1 distribution mechanism involves at least partial entry of the Nfs1 precursor into mitochondria, and then retrieval of a minor subpopulation (probably by reverse translocation) into the cytosol and then the nucleus. To further elucidate the mechanism of Nfs1 distribution we determined the N-terminal mitochondrial sequence of Nfs1 by Edman degradation. This led to the discovery of a novel mitochondrial processing enzyme, Icp55. This enzyme removes three amino acids from the N terminus of Nfs1 after cleavage by mitochondrial processing peptidase. Intriguingly, Icp55 protease (like its substrate Nfs1) appears to be dual distributed between the nucleus and mitochondria.Eclipsed distribution is a specific case of the dual protein subcellular localization in which one compartment contains the vast majority of a specific protein while a second compartment contains only a minute (eclipsed) amount (1-3). Therefore, eclipsed distributed proteins are hard to detect, and it is hard to discriminate between the specific functions of each isoprotein. For example, aconitase, a well characterized mitochondrial protein that participates in the tricarboxylic acid cycle, has been recently shown to be present in minute amounts in the cytosol, where it is crucial for the function of the glyoxylate shunt (2, 3).The protein NifS was first identified and characterized in Azotobacter vinelandii, as part of the nitrogen fixation (nif) operon (4). This enzyme is a pyridoxal 5Ј-phosphate-dependent cysteine desulfurase. In eukaryotes, an NifS-like protein termed Nfs1 has been identified, which shows a highly conserved sequence homology. The Saccharomyces cerevisiae mitochondrial Nfs1 has been shown to participate in iron-sulfur cluster assembly (5). Nakai and coworkers identified an NLS 5 -like sequence, RRRPR, in the mature sequence of yeast Nfs1, that is highly conserved in eukaryotic Nfs1 proteins (e.g. in yeast, mouse, and human) (6). When this NLS-like sequence was mutated to RRGSR, the mutant gene could not complement cell growth of a chromosomal NFS1-depleted strain. Nevertheless, this mutation did not affect the function of Nfs1 in the biogenesis of iron-sulfur proteins (5, 6). Nfs1 performs an additional function in the post-transcriptional modification of mitochondrial and cytosoli...

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Section: Nfs1 Is Processed By Two Peptidases Mpp and The Novel Icp55-mentioning

confidence: 99%

Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

Naamati

Regev‐Rudzki

Galperin

et al. 2009

Journal of Biological Chemistry

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“…Analogous to SUS1 or SH1, adenylate kinase is predominantly cytosolic, with a minor fraction (only up to 6 -10%) distributed also in mitochondria (48,49). Adenylate kinase has an internal targeting sequence that is critical for organellar import (48). SUS1 possesses a putative targeting sequence very close to the N terminus (residues 8 -72).…”

Section: Sus Is An Integral Protein Of Maize Mitochondria-mentioning

confidence: 99%

“…Mtf1p) are imported by mitochondria despite lacking any known targeting information at their N termini (41,47). Analogous to SUS1 or SH1, adenylate kinase is predominantly cytosolic, with a minor fraction (only up to 6 -10%) distributed also in mitochondria (48,49). Adenylate kinase has an internal targeting sequence that is critical for organellar import (48).…”

Section: Sus Is An Integral Protein Of Maize Mitochondria-mentioning

confidence: 99%

Mitochondrial Localization and Putative Signaling Function of Sucrose Synthase in Maize

Subbaiah

Palaniappan

Duncan

et al. 2006

Journal of Biological Chemistry

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In many organisms, an increasing number of proteins seem to play two or more unrelated roles. Here we report that maize sucrose synthase (SUS) is distributed in organelles not involved in sucrose metabolism and may have novel roles beyond sucrose degradation. Bioinformatics analysis predicts that among the three maize SUS isoforms, SH1 protein has a putative mitochondrial targeting peptide (mTP). We validated this prediction by the immunodetection of SUS in mitochondria. Analysis with isoform-specific antisera revealed that both SH1 and SUS1 are represented in mitochondria, although the latter lacks a canonical mTP. The SUS2 isoform is not detectable in mitochondria, despite its presence in the cytosol. In maize primary roots, the mitochondrion-associated SUS (mtSUS; which includes SH1 and SUS1) is present mostly in the root tip, indicating tissue-specific regulation of SUS compartmentation. Unlike the glycolytic enzymes that occur attached to the outside of mitochondria, SH1 and SUS1 are intramitochondrial. The low abundance of SUS in mitochondria, its high K m value for sucrose, and the lack of sucrose in mitochondria suggest that mtSUS plays a non-sucrolytic role. Co-immunoprecipitation studies indicate that SUS interacts with the voltage-dependent anion channel in an isoform-specific and anoxia-enhanced manner and may be involved in the regulation of solute fluxes into and out of mitochondria. In several plant species, at least one of the SUS proteins possesses a putative mTP, indicating the conservation of the noncatalytic function across plant species. Taken together, these observations suggest that SUS has a novel noncatalytic function in plant cells.In both plant and animal cells, a small number of catalytic and structural proteins play additional roles that in some cases are regulatory in nature (1-3). These "moonlighting" proteins possess the ability to assemble into multiprotein complexes and mediate sophisticated biological functions such as integrating signals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2 is the most studied among such versatile proteins (4). Besides its pivotal role in energy metabolism, GAPDH is shown to be involved in membrane fusion, microtubule assembly, RNA transport, DNA replication and repair, and cell death (5). The functional diversity of GAPDH is facilitated by its ability to interact with other proteins and translocate to multiple subcellular compartments. Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose and is a key player in plant sucrose catabolism. In maize, the following three genes encoding sucrose synthase are known: sus1, sus2, and sh1. The isoforms encoded by these genes are symbolized SUS1, SUS2, and SUS-SH1, respectively (6). In this study, for the sake of simplicity, we call the SUS-SH1 isoform SH1. All the three isoforms are predominantly recovered as soluble (cytosolic) proteins from plant cells, in accordance with their predicted secondary structures. The well documented function of this en...

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“…As a loading control, we used antibodies to detect Aky2p [18], a protein which is stable throughout the cell cycle and highly resistant to proteolytic degradation [40].…”

Section: Cell-cycle Arrest Experiments To Test Rio1p Stabilitymentioning

confidence: 99%

“…Logarithmically growing cells were induced by galactose (RIO1 alleles under the control of the GAL10 promoter) and simultaneously arrested by treatment with either a-factor (arrest before the G 1 ⁄ S transition), hydroxyurea (S phase), or nocodazole (before onset of anaphase), respectively. Cellular concentrations of Rio1p were measured relative to adenylate kinase 2 (Aky2p), which is a constitutively expressed, stable protein [18] as a loading control (see Experimental procedures). Our results indicate that in the RIO1 wild-type and the (S > D) 6 mutant the level of Rio1 protein is low to undetectable in the S phase but normal in G 1 and during mitosis compared with cycling cells ( Fig.…”

Section: Biological Implications Of Phosphorylation Of Rio1p By Ck2mentioning

confidence: 99%

Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell‐cycle phase‐dependent degradation in yeast

et al. 2007

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The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotes and is responsible for the Ser ⁄ Thr phosphorylation of a large number of protein substrates [1][2][3]. The active holoenzyme is most often a heterotetramer composed of two catalytic a subunits, a (encoded by CKA1) and a¢ (encoded by CKA2), and two regulatory b subunits, b and b¢ in Saccharomyces cerevisiae (CKB1 and CKB2). The enzyme occurs in all possible combinations of a and b subunits [4,5]. In yeast, deletion of the gene for one of the two catalytic subunits has little effect, but deletion of both homologous genes results in loss of viability [6]. To date, more than 300 endogenous CK2 substrates are known to be involved in quite diverse processes, e.g. cell proliferation, signal transduction, transcriptional regulation, translation and metabolism [3]. Despite this eminent role in strictly regulated cellular processes and although CK2 is indispensable for cell life, CK2 activity by itself is apparently unregulated [4,5], although some fluctuation in activity in correlation with cell-cycle progression has been seen in cultured mammalian cells [7,8]. The physiological effect of substrate phosphorylation is surprisingly low with almost all targets described to date [9,10]. In S. cerevisiae, it has been shown using temperature-sensitive CKA2 alleles that protein kinase CK2 is required for passage across the G 2 ⁄ M boundary and for cell-cycle progression through the G 1 phase [11]. Using co-immunoprecipitation combined with MS analysis, we identified the a¢ subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Dcka1 or Dcka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A) 6 mutant proteins. Both mutant alleles (S > A) 6 or (S > D) 6 complement in vivo, however, growth of the RIO1 (S > A) 6 mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D) 6 mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G 1 ⁄ S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.Abbreviations Aky2p, adenylate kinase 2; CK2, casein kinase 2; GST, glutathione S-transferase.

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Redundant Mitochondrial Targeting Signals in Yeast Adenylate Kinase

Cited by 18 publications

References 59 publications

Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

Mitochondrial Localization and Putative Signaling Function of Sucrose Synthase in Maize

Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell‐cycle phase‐dependent degradation in yeast

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