Functional analysis of human mitochondrial receptor Tom20 for protein import into mitochondria

The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106-125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitro import of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.     

Mrs5p, an essential protein of the mitochondrial intermembrane space, affects protein import into yeast mitochondria

We have isolated a yeast nuclear gene that suppresses the previously described respiration-deficient mrs2-1 mutation when present on a multicopy plasmid. Elevated gene dosage of this new gene, termed MRS5, suppresses also the pet phenotype of a mitochondrial splicing-deficient group II intron mutation M1301. The MRS5 gene product, a 13-kDa protein of low abundance, shows no similarity to other known proteins and is associated with the inner mitochondrial membrane, protruding into the intermembrane space. MRS5 codes for an essential protein, as the disruption of this gene is lethal even during growth on fermentable carbon sources. Thus, the Mrs5 protein seems to be involved in mitochondrial key functions aside from oxidative energy conservation, which is dispensable in fermenting yeast cells. Depletion of Mrs5p in yeast cells causes accumulation of unprocessed precursors of the mitochondrial hsp60 protein and defects in all cytochrome complexes. These findings suggest an essential role of Mrs5p in mitochondrial biogenesis.     

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.     

Co-translational protein import into mitochondria: an alternative view

Recent in vitro and in vivo experiments suggest that the synthesis and import of mitochondrial proteins are very tightly coupled and that a co-translational import reaction may be mandatory for some proteins. These results are entirely consistent with early experiments which suggested that import occurs co-translationally and that cytosolic polysomes synthesizing mitochondrial proteins are bound to protein import sites on isolated mitochondria. This article discusses and seemingly contradictory reports concerning the involvement of co-translational and post-translational mechanisms in the import process and examines the impact of recent developments in the field.     

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.     

The yeast nascent polypeptide-associated complex initiates protein targeting to mitochondria in vivo

The yeast nascent polypeptide-associated complex (NAC) is encoded by two genes, EGD1 and EGD2, and is associated with cytoplasmic ribosomes. Yeast mutants lacking NAC (Deltaegd2) are viable but suffer slight defects in the targeting of nascent polypeptides to several locations including the endoplasmic reticulum and mitochondria. If both NAC and Mft52p are missing from yeast cells, inefficient targeting of mitochondrial precursor proteins leads to defects in both mitochondrial function and morphology. We suggest that NAC provides a ribosomal environment for nascent mitochondrial targeting sequences to achieve secondary structure, thereby enhancing the efficiency of protein targeting.     

Transportin: nuclear transport receptor of a novel nuclear protein import pathway

The main purposes of this study were to investigate the best parameter for describing gallbladder emptying and whether gallbladder bile emptying should be induced with a bolus injection or continuous infusion of cholecystokinin-octapeptide (CCK-8). METHODS: Gallbladder emptying was measured by dynamic cholescintigraphy. Twelve healthy subjects and six patients with gallstones were examined twice with CCK-8 infusion cholescintigraphy, 0.3 ng CCK-8 kg per min for 60 min under identical circumstances. Another six healthy subjects randomly received bolus injection (0.04 microgram/kg) and infusion of CCK-8 (0.3 ng/kg per min for 60 min), respectively, during cholescintigraphy on two separate occasions. The choice of bolus dose was based on recommendations from the CCK-8 manufacturer. The infusion dose was chosen to produce plasma CCK concentrations similar to postprandial plasma CCK levels. RESULTS: A parameter of gallbladder emptying, mean ejection fraction (EF), was defined as 100% minus the area under the time-activity curve normalized to 100% and divided by the time interval from maximum to minimum counts per minute. This parameter proved superior to the well known parameters, EFmax. and EF30, in regard to reproducibility in healthy subjects. The slope of the regression line for the mean EF was 0.998 and the intercept value approximately 0% (p = 0.0001). The mean coefficient of variation was 4%. Apart from a higher mean coefficient of variation, similar reproducibility results were seen in the six patients. The measurements of EF30 in healthy subjects scattered more widely around the mean compared to the mean EF and EFmax, which indicates poorer ability to separate normal from abnormal gallbladder emptying. Intravenous bolus injection of CCK-8 resulted in incomplete gallbladder emptying with a mean EF value of 16% (s.d. 9%; range 7%-32%) compared to 49% (s.d. 7%; range 37%-57%) following CCK-8 infusion (p = 0.004). Abdominal discomfort was observed in all subjects after administration of the bolus injection, whereas no complaints were reported during infusion. CONCLUSION: Mean EF is the best parameter for describing gallbladder emptying. Moreover, slow infusion of a physiological dose of CCK-8 is preferable to induce gallbladder emptying because it results in more complete emptying and has no side effects.     

A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris

Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Delta mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Delta cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Delta mutant and were impaired in binding to the PTS2 sequence.     

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1 beta subunit of the ATP synthase (pF1 beta) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondrial than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1 beta with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1 beta into intact mitochondria. Import of pF1 beta through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.     

Cytochrome c heme lyase activity of yeast mitochondria

A highly efficient in vitro system was established for measuring by high performance liquid chromatography the formation of holocytochrome c by yeast mitochondria. Holocytochrome c formation required reducing agents, of which dithiothreitol was the most effective. With biosynthetically made, pure Drosophila melanogaster apocytochrome c and Saccharomyces cerevisiae mitochondria, the activity of cytochrome c heme lyase amounted to about 800 fmol min-1 mg-1 mitochondrial protein. The kinetics were typical Michaelis-Menten (Km approximately 1 nM), as were those of mitoplasts with broken outer membranes (Km approximately 3 nM). As tested with mitoplasts, holocytochromes c from a range of species were found to be competitive inhibitors of heme lyase at physiological concentrations, providing a mechanism for controlling this concentration in vivo. Apocytochrome c associated with yeast mitochondria in two phases of Kd approximately 2 x 10(-10) and 10(-8) M, respectively, whereas mitoplasts had lost the high affinity binding. A site-directed mutant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, glutamic acid, and asparagine) was found to be converted to holocytochrome c (Km approximately 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding. Thus, the high affinity binding of apocytochrome c to mitochondria is not directly related to holocytochrome c formation.     

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria

Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.     

The aminoacceptor stem of the yeast tRNA(Lys) contains determinants of mitochondrial import selectivity

The effect of quinine, a cinchona alkaloid, was studied on gastrointestinal transit in mice. Intraperitoneal (i.p.) administration of quinine inhibited the intestinal propulsion of a charcoal suspension at a dose of 100 mg/kg, comparing favorably with 5 mg/kg morphine. In an attempt to probe into the mechanism underlying this inhibition, a possible modulation by minoxidil (1 mg/kg, p.o.) and glibenclamide (1 mg/kg, p.o.), the drugs that, respectively, open and close ATP-sensitive K+ channels was tested on gastrointestinal transit in animals treated or not with quinine or morphine. While minoxidil produced no significant change of normal transit, glibenclamide significantly increased it. However, both drugs blocked the quinine-induced reduction in gastrointestinal transit. In contrast, the inhibitory effect of morphine on gastrointestinal transit was not modified by either drug. The effects of quinine as well as of morphine on gastrointestinal transit were significantly antagonized by naloxone (2 mg/kg, s.c.), a mu-opioid receptor antagonist but not by yohimbine (1 mg/kg, i.p.), an alpha2-adrenoceptor antagonist. Furthermore, quinine at a lower dose (25 mg/kg) that showed no per se effect on gastrointestinal transit, significantly potentiated the response to 2.5 mg/kg morphine. Although the role of ATP-sensitive K+ channels in the action of quinine and morphine was not clarified by the present results, a possible involvement of endogenous opioid(s) in the quinine-induced inhibition of gastrointestinal transit can be suggested.     

Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA.     

Nucleocytoplasmic shuttling by protein nuclear import factors

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

A novel receptor-mediated nuclear protein import pathway

Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.     

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5

Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70. We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.     

The import route of ADP/ATP carrier into mitochondria separates from the general import pathway of cleavable preproteins at the trans side of the outer membrane

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.