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.     

Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis

Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes. StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence. We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E. coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import.     

Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.     

The multiligand-binding protein gC1qR, putative C1q receptor, is a mitochondrial protein

A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.     

cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells

Mitogen-activated protein kinases (MAPKs) are activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors. Using transient transfection of COS-7 cells, we have studied the stimulation of a hemagglutinin-tagged p44mapk (p44HA-mapk) by receptors coupled to Gs, Gq, and Gi. Agonists that act via all three G proteins stimulated p44HA-mapk activity. A constitutively activated alpha s mutant, forskolin, and a cAMP analog also increased p44HA-mapk activity, indicating that cAMP in COS-7 cells, in contrast to other cell types, activates the MAPK pathway. Similarly, a constitutively activated alpha q mutant, overexpression of phospholipase C-beta 2, and a phorbol ester also stimulated p44HA-mapk, suggesting that Gq-coupled receptors stimulate the MAPK pathway by increasing phosphatidylinositol turnover and probably stimulating protein kinase C. In COS-7 cells, in contrast to Rat-1 cells, mutationally activated alpha i did not stimulate the MAPK pathway. G protein beta and gamma subunits, overexpressed together, did activate p44HA-mapk; this finding suggests that in COS-7 cells Gi-coupled receptors may stimulate the MAPK pathway through beta gamma. These unexpected results in COS-7 cells show that G proteins and second messengers regulate the MAPK pathway differently in different cell types.     

Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.     

ATP and a mitochondrial electrochemical gradient are required for functional activity of the steroidogenic acute regulatory (StAR) protein in isolated mitochondria

The Steroidogenic Acute Regulatory (StAR) protein has been put forth as the rapidly synthesized, cycloheximide-sensitive protein that is required for the transport of cholesterol to the inner mitochondrial membrane and the P450scc enzyme and thereby acutely regulates steroidogenesis in steroidogenic tissues. In this study, several of the factors that may be required for StAR activity were examined using an in vitro system. Lysates from StAR-transfected COS-1 cells were added to mitochondria isolated from MA-10 Leydig tumor cells. Results obtained demonstrated that StAR-containing cell lysate increased steroidogenesis in isolated mitochondria, but failed to do so in the presence of m-CCCP, apyrase, or AMP-PNP, suggesting that StAR function requires ATP hydrolysis as well as an electrochemical gradient for maximal steroidogenic activity.     

Increasing DNA repair capacity in bone marrow by gene transfer as a prospective tool in cancer therapy

The Rh50 glycoprotein is suspected of being involved in Rh antigen expression. We prepared Rh50 cDNA from a human bone marrow library by polymerase chain reaction and then subcloned this cDNA into various vectors. The vector containing Rh50 cDNA produced a 30-kDa nonglycosylated form of Rh50 in a rabbit reticulocyte lysate system and produced partially glycosylated Rh50 (32 kDa) when microsomes were added to the system. COS-1 cells transiently transfected with the vector containing Rh50 cDNA produced partially glycosylated Rh50 (32 kDa) recognized by a Rh50-specific antibody. Surface expression of Rh50 in K562 cells was also detected by flow cytometry using mouse monoclonal antibody (2D10) specific to Rh50. Partially glycosylated Rh50 (32 kDa) was again isolated from the lysates of K562 cells metabolically labeled with [35S]-methionine or [3H]-mannose using anti-Rh50 antisera. These systems (K562 and COS-1 cells) should prove useful for studying the transport of Rh proteins within the cell and the necessary components needed for Rh antigenicity at the cell surface.     

Collagen injection therapy for post-prostatectomy incontinence

Serial deletions of the N-terminal 319 amino acids of rPLD1 expressed in COS-7 cells resulted in increased basal PLD activity. Incubation of the cells with phorbol myristate acetate increased the activity of endogenous and wild-type rPLD1. The mutant rPLD1 with deletion of the first 50 amino acids responded to the phorbol ester, however, rPLD1 with deletions of 115 amino acids or more did not. In cells in which constitutively active V14RhoA was co-expressed with the mutant PLDs, stimulation of PLD activity was observed with all deletion mutants. In membranes from COS-7 cells in which the mutant PLDs were expressed, only the mutant with deletion of 50 N-terminal amino acids responded to added protein kinase C-alpha and phorbol ester, in agreement with the in vivo studies. When myristoylated ADP-ribosylation factor 3 (mARF3) was added together with guanosine 5'-3-O-(thio)triphosphate, all mutants showed stimulation of PLD activity. It is concluded that the site of interaction of protein kinase C with rPLD1 is located in the N-terminal region and that Rho and ARF interact at other sites.     

Intracellular retention of the corticotrophin-releasing hormone (CRH) precursor within COS-7 cells

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.     

Identification and characterization of a human protein kinase related to budding yeast Cdc7p

The Cdc7p protein kinase is essential for the G1/S transition and initiation of DNA replication during the cell division cycle in Saccharomyces cerevisiae. Cdc7p appears to be an evolutionarily conserved protein, since a homolog Hsk1 has been isolated from Schizosaccharomyces pombe. Here, we report the isolation of a human cDNA, HsCdc7, whose product is closely related in sequence to Cdc7p and Hsk1. The HsCdc7 cDNA encodes a protein of 574 amino acids with predicted size of 64 kDa. HsCdc7 contains the conserved subdomains common to all protein-serine/threonine kinases and three "kinase inserts" that are characteristic of Cdc7p and Hsk1. Immune complexes of HsCdc7 from cell lysates were able to phosphorylate histone H1 in vitro. Indirect immunofluorescence staining demonstrated that HsCdc7 protein was predominantly localized in the nucleus. Although the expression levels of HsCdc7 appeared to be constant throughout the cell cycle, the protein kinase activity of HsCdc7 increased during S phase of the cell cycle at approximately the same time as that of Cdk2. These results, together with the functions of Cdc7p in yeast, suggest that HsCdc7 may phosphorylate critical substrate(s) that regulate the G1/S phase transition and/or DNA replication in mammalian cells.     

Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.     

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.     

Calcium sensitization in perfused beating guinea pig heart by a positive inotropic agent MCI-154

An in vitro import system was used to characterize the mechanism of import of phospholipid hydroperoxide glutathione peroxidase (PHGPx) into mitochondria. Mitochondria were isolated from rat liver and incubated at 25 degrees C with [35S]methionine-labeled products of the in vitro translation of mRNA that encoded 23-kDa and 20-kDa PHGPx. 23-kDa PHGPx was imported into mitochondria in a time-dependent manner and was processed to yield the 20-kDa form of PHGPx. The 20-kDa form of PHGPx, without a leader sequence, associated weakly with mitochondria but was not imported. An analysis with an uncoupler of oxidative phosphorylation showed that a membrane potential in the mitochondria was also required for the import of PHGPx. It appears, therefore, that the leader sequence in the precursor to PHGPx is the signal for import into the mitochondria. This is the first report to indicate that the precursor to PHGPx is imported into the mitochondria via the action of a leader sequence.     

A new meiotic endonuclease from Coprinus meiocytes

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.     

Cloning and deduced amino acid sequence of a novel cartilage protein (CILP) identifies a proform including a nucleotide pyrophosphohydrolase

The cDNA cloning and expression in vitro and in eukaryotic cells of a novel protein isolated from human articular cartilage, cartilage intermediate layer protein (CILP) is described. A single 4. 2-kilobase mRNA detected in human articular cartilage encodes a polypeptide of 1184 amino acids with a calculated molecular mass of 132.5 kDa. The protein has a putative signal peptide of 21 amino acids, and is a proform of two polypeptides. The amino-terminal half corresponds to CILP (molecular mass of 78.5 kDa, not including post-translational modifications) and the carboxyl-terminal half corresponds to a protein homologous to a porcine nucleotide pyrophosphohydrolase, NTPPHase (molecular mass of 51.8 kDa, not including post-translational modifications). CILP has 30 cysteines and six putative N-glycosylation sites. The human homolog of porcine NTPPHase described here contains 10 cysteine residues and two putative N-glycosylation sites. In the precursor protein the NTPPHase region is immediately preceded by a tetrapeptide conforming to a furin proteinase cleavage consensus sequence. Expression of the full-length cDNA in a cell-free translation system and in COS-7 or EBNA cells indicates that the precursor protein is synthesized as a single polypeptide chain that is processed, possibly by a furin-like protease, into two polypeptides upon or preceding secretion.     

Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells

We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.