Import of an incompletely folded precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP.

We have studied the post-translational import of incomplete precursor chains into isolated yeast mitochondria. The precursor was a fusion protein containing a mitochondrial presequence attached to mouse dihydrofolate reductase. In vitro-synthesis of the precursor was interrupted by the elongation inhibitor cycloheximide and the arrested nascent chains cosedimenting with ribosomes were released by EDTA. These incomplete chains were efficiently imported by isolated yeast mitochondria; their import resembled that of the complete precursor in requiring an energized inner membrane and a mitochondrial presequence. It differed from that of the completed precursor in its resistance to methotrexate (which only binds to correctly folded dihydrofolate reductase) and its independence of added ATP. The incomplete chains were also more sensitive to proteinase K than the completed precursor. We conclude that the incomplete chains were incompletely folded and suggest that the lack of tight folding caused import into mitochondria to become independent of added ATP. This implies that ATP may participate, directly or indirectly, in the unfolding of the precursor for its transport into mitochondria.     

The entire N-terminal half of TatC is involved in twin-arginine precursor binding

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.     

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.     

Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported

The thylakoid t win a rginine protein t ranslocation (Tat) system is thought to have a multivalent receptor complex with each cpTatC‐Hcf106 pair constituting a signal peptide‐binding unit. Conceptual models suggest that translocation of individual precursor proteins occurs upon assembly of a Tha4 oligomer with a precursor‐occupied cpTatC‐Hcf106. However, results reported here reveal that multiple precursor proteins bound to a single receptor complex can be transported together. Precursor proteins that contain one or two cysteine residues readily formed intermolecular disulphide bonds upon binding to the receptor complex, resulting in dimeric and tetrameric precursor proteins. Three lines of evidence indicate that all members of precursor oligomers were specifically bound to a receptor unit. Blue native–polyacrylamide gel electrophoresis analysis showed that oligomers were present on individual receptor complexes rather than bridging two or more receptor complexes. Upon energizing the membrane, the dimeric and tetrameric precursors were transported across the membrane with efficiencies comparable with that of monomeric precursors. These results imply a novel aspect of Tat systems, whereby multiple precursor‐binding sites can act in concert to transport an interlinked oligo‐precursor protein.     

Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development

The murine epidermal growth factor (EGF) precursor is a 1217 amino acid protein which contains mature EGF (amino acid residues 977-1029) as well as eight EGF-like repeats. Although the highest levels of EGF are found in the adult male mouse submandibular gland, the results of in situ hybridization studies and mRNA analyses suggest that EGF precursor mRNA is synthesized in several adult mouse tissues including the lung and the incisor. To determine if EGF precursor gene expression is intrinsic to the developmental program for either embryonic tooth or lung organogenesis, sense and antisense oligodeoxyribonucleotide probes corresponding to amino acids 1070-1081 of the precursor were used to localize cellular sites of synthesis of EGF precursor mRNA by in situ hybridization. Antibodies directed against amino acid residues 348-691 of the precursor were used in immunodetection techniques to identify either EGF precursor protein or processed derivatives. In contrast to earlier reports indicating that embryonic mouse tissues do not synthesize EGF precursor mRNA, we found that EGF precursor mRNA is present in clusters of ectoderm-, mesoderm-, and ectomesenchyme-derived cells associated with embryonic teeth and lung organs. Moreover, epitopes common to the EGF precursor were immunolocalized in both the epithelial and mesenchymal tissues of embryonic mouse tooth and lung organs. These results suggest that the EGF precursor and/or motifs contained within the precursor molecule, including mature EGF, may play an instructive or permissive role in epithelial-mesenchymal interactions pursuant to organogenesis.     

Cathepsin D precursors in clathrin-coated organelles from human fibroblasts

Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.     

Endotoxic properties of synthetic pentaacyl lipid A precursor Ib and a structural isomer

A pentaacyl precursor of lipid A biosynthesis, termed precursor Ib, and a structural isomer have been chemically synthesized. These compounds were, in comparison to synthetic Escherichia-coli type lipid A or lipopolysaccharide, analyzed for their activity in typical endotoxin test systems. It was found that both precursor Ib and the isomer exhibited similar or only slightly lower pyrogenic, lethal and Shwartzman-phenomenon-inducing activity than lipid A. All preparations were comparable in their B-lymphocyte mitogenicity, macrophage-activating capacity and immunoreactivity towards lipid A antisera. The proton nuclear magnetic resonance spectra of the 1-dephospho derivative of synthetic and bacterial precursor Ib were indistinguishable proving that the previously proposed structure for precursor Ib is correct.     

Effect of reduced membrane lipid fluidity on the biosynthesis of lipopolysaccharide of Escherichia coli

A low molecular weight precursor of lipopolysaccharide was accumulated under conditions in which the membrane lipids of a fatty acid auxotroph of Escherichia coli were reduced to a non-fluid state. The lipopolysaccharide precursor was detected, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography, in membranes isolated from cells which were pulse-labeled with N-acetyl-[1-14C]glucosamine. The precursor could be chased into mature lipopolysaccharide by returning the membrane lipids to a normal fluid state. Conversion of the precursor to lipopolysaccharide was inhibited by the presence of potassium cyanide or sodium arsenate. The processing of several outer membrane protein precursors, including the promatrix proteins, was also inhibited under these conditions. Preliminary characterization of the lipopolysaccharide precursor was undertaken.     

Association of the precursor of cathepsin D with coated membranes. Kinetics and carbohydrate processing

Specific anti-chlathrin antibodies were used to isolate clathrin-coated membranes from homogenates of metabolically labelled fibroblasts. The isolated membranes were extracted with detergents and cathepsin D was isolated from the extracts. The 53-kDa precursor of cathepsin D was transiently associated with the coated membranes with a maximum approximately 60 min after synthesis. At maximum about 4.0% of the precursor was recovered with the coated membranes and the associated precursor contained complex oligosaccharides. The proportion of complex oligosaccharides in the coated membrane-associated precursor did not differ from that in the total precursor. These data support the view that coated membranes are involved in the transport of cathepsin D between trans Golgi and a prelysosomal organelle.     

Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.     

Different transforming growth factor-alpha species are derived from a glycosylated and palmitoylated transmembrane precursor

cDNA analysis has revealed that the 50 amino acid transforming growth factor-alpha (TGF-alpha) is derived from a 160 amino acid precursor. Antibodies to TGF-alpha and to a C-terminal portion of the precursor were used to study the biosynthesis and processing of the precursor. CHO cells transfected with a TGF-alpha expression vector secrete high levels of TGF-alpha; a mixture of species of about 18 kd is secreted in addition to the 50 amino acid form. These larger species are N-glycosylated and are derived from the same precursor as the smaller form. The C-terminal segment of the precursor remains anchored in the membrane and has covalently attached palmitate. The newly synthesized TGF-alpha precursor is thus a transmembrane protein that subsequently undergoes external proteolytic cleavages, releasing several TGF-alpha species.     

Translocation of proteins into rat liver mitochondria. The precursor polypeptides of a large subunit of succinate dehydrogenase and ornithine aminotransferase and their imports into their own locations of mitochondria

The precursor polypeptides of a large subunit of succinate dehydrogenase and ornithine aminotransferase (the enzymes which are located in the mitochondrial inner membrane and matrix respectively) were synthesized as a larger molecular mass than their mature subunits, when rat liver RNA was translated in vitro. These precursor polypeptides were also detected in vivo in ascites hepatoma cells (AH-130 cells). When the 35S-labeled precursor polypeptides were incubated with isolated rat liver mitochondria at 30 degrees C in the presence of an energy-generating system, these two precursors were converted to their mature size and the 35S-labeled mature-size polypeptides associated with mitochondria. Furthermore, these mature-size polypeptides were recovered from their own locations, the inner mitochondrial membrane and the matrix. The precursor of ornithine aminotransferase incubated with rat liver mitochondria at 0 degree C was specifically and tightly bound to the surface of the mitochondria even in the presence of an uncoupler of oxidative phosphorylation. This precursor, bound to the mitochondria, was imported into the matrix when the mitochondria were reisolated and incubated at 30 degrees C in the presence of an energy-generating system, suggesting that a specific receptor may be involved in the binding of the precursor. The processing enzyme for both precursor polypeptides seemed to be located in the mitochondrial matrix and was partially purified from the mitochondria. A metal-chelating agent strongly inhibited the processing enzyme and the inhibition was recovered by the addition of Mn2+ or Co2+.     

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.     

Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria.

In order to study the role of protein unfolding during post-translational protein import into mitochondria, we destabilized the structure of a mitochondrial precursor protein by site-directed mutagenesis. The precursor consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor fused to mouse dihydrofolate reductase. Labilization of the folded precursor structure was monitored by increased susceptibility to protease and diminished ability of methotrexate to block import of the precursor into isolated yeast mitochondria. On comparing the original precursor with two mutant forms that were destabilized to different degrees, increased labilization correlated with an increased rate and efficiency of import into mitochondria. This supports the view that the precursor must unfold in order to enter the mitochondria.     

Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.     

The β-Amyloid Precursor Protein (APP) and Alzheimer's Disease: Does the Tail Wag the Dog?

The β-amyloid precursor protein has been the focus of much attention from the Alzheimer's disease community for the past decade and a half. The β-amyloid precursor protein holds a pivotal position in Alzheimer's disease research because it is the precursor to the amyloid β-protein which many believe plays a central role in Alzheimer's disease pathogenesis. It was also the first gene in which mutations associated with inherited Alzheimer's disease were found. Although the molecular details of the generation of amyloid β-protein from β-amyloid precursor protein are being unraveled, the actual physiological functions of β-amyloid precursor protein are far from clear. This situation is changing as accumulating new evidence suggests that the C-terminal cytosolic tail of β-amyloid precursor protein may have multiple biological activities, ranging from axonal transport to nuclear signaling. This article reviews the current state of knowledge about the biological functions of β-amyloid precursor protein .     

Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci

Cytoplasmic precursors of the peptidoglycan biosynthetic pathway were purified from vancomycin-treated, glycopeptide-sensitive and -resistant strains of Enterococcus faecium. Resistance was due to production of a modified precursor, UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate, where lactate was identified on the basis of mass of the precursor and on its ability to act as a substrate for D-lactate dehydrogenase after release from the precursor. The presence of the D-lactate residue instead of D-alanine in the terminal position would hinder formation of a vancomycin-precursor complex, without preventing incorporation of the precursor into mature peptidoglycan.     

Post-translational processing of bovine chondromodulin-I

Chondromodulin-I (ChM-I) is a small glycoprotein that is abundant in fetal cartilage. Mature chondromodulin-I is processed from a larger precursor form, presumably at a proteolytic site RERR-ELVR. The precursor, mature chondromodulin-I and two processed products, the remnant left after removal of mature chondromodulin-I and a smaller, unglycosylated form, were identified using antipeptide antisera. The products of chondromodulin-I precursor processing were seen in cultured chondrocytes, a stable long-term culture chondrosarcoma cell line, as well as Chinese hamster ovary (CHO) cells transfected with an expression plasmid that contained cDNA coding for the chondromodulin-I precursor. Pulse-chase analysis allowed a processing pathway to be analyzed for chondromodulin-I. To further dissect the processing events, three constructs that express recombinant wild-type or mutant chondromodulin-I were transfected into CHO cells. We showed that chondromodulin-I is cleaved intracellularly at the predicted cleavage site, and that the mature glycopeptide is rapidly secreted immediately after processing. The chondromodulin-1 precursor has a short half-life and is not readily apparent in tissue samples, suggesting that chondromodulin is not a member of the juxtacrine family of growth factors, despite some similarities. The smaller unglycosylated form of chondromodulin-I was only observed in cartilage and not in short-term cultures or transfected cells, suggesting an extracellular processing event. No processing occurred when the precursor cleavage site was mutated to RERQ-SLVR or when precursor chondromodulin-I was expressed in the furin-deficient CHO cell line, suggesting the involvement of furin in processing.     

The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly

Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.     

A conformationally altered precursor to the lysosomal enzyme alpha-mannosidase accumulates in the endoplasmic reticulum in a mutant strain of Dictyostelium discoideum

The mutant strain of Dictyostelium discoideum, HMW-437, contains a mutation in the structural gene coding for the lysosomal enzyme alpha-mannosidase. Unlike the wild type strain, Ax3, this strain fails to proteolytically process or secrete the 140,000-dalton alpha-mannosidase precursor. The level of sulfate incorporation into the mutant precursor was significantly lower when compared to the wild type precursor. In addition, the mutant precursor was entirely sensitive to endoglycosidase H. Subcellular fractionation of HMW-437 membranes indicated that the majority of the alpha-mannosidase precursor sedimented in a region of the gradient corresponding to the rough endoplasmic reticulum. This accumulation within the rough endoplasmic reticulum did not appear to result from gross conformational changes which lead to aggregation. Trypsin digestion of radioactively labeled Ax3 and HMW-437 precursors demonstrated that there were differences in susceptibility to protease cleavage between the wild type and mutant alpha-mannosidase precursor molecules, suggesting that a minor conformational change could contribute to the accumulation of the mutant precursor inside the endoplasmic reticulum.