Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase

Translocation of proteins across membranes of the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in the transported proteins. To test whether the transport of proteins into peroxisomes is also mediated by a peptide targeting signal, we have studied the firefly luciferase gene that encodes a protein transported to peroxisomes in both insect and mammalian cells. We have identified two regions of luciferase which are necessary for transport of this protein into peroxisomes. We demonstrate that one of these, region II, represents a peroxisomal targeting signal because it is both necessary and sufficient for directing cytosolic proteins to peroxisomes. The signal is no more than twelve amino acids long and is located at the extreme carboxy-terminus of luciferase. The location of the targeting signal for translocation across the peroxisomal membrane therefore differs from the predominantly amino-terminal location of signals responsible for transport across the membranes of the endoplasmic reticulum, chloroplast, or mitochondrion.     

Tight folding of a passenger protein can interfere with the targeting function of a mitochondrial presequence.

The first seven residues of the yeast cytochrome oxidase subunit IV presequence are insufficient to target attached mouse dihydrofolate reductase into isolated yeast mitochondria. However, the targeting function of this truncated presequence can be restored by presenting the fusion protein to isolated mitochondria either as nascent, unfolded chains, or as full-length chains whose dihydrofolate reductase moiety had been destabilized either by urea treatment or by point mutations. The targeting efficiency of a mitochondrial presequence can thus be strongly influenced by the conformation of the attached 'passenger protein'. These results also underscore the difficulty of defining a 'minimal' mitochondrial targeting signal.     

Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins

As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.     

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.     

Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.     

Escherichia coli TonB protein is exported from the cytoplasm without proteolytic cleavage of its amino terminus

The requirement for TonB protein in a variety of membrane-related processes suggests that TonB is an envelope protein. Consistent with this suggestion, the deduced TonB amino acid sequence (Postle, K., and Good, R. F., (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5235-5239) contains an amino-terminal region similar to leader (signal) sequences of exported proteins, although its charged region falls outside the rules which characterize these sequences (von Heijne, G. (1985) J. Mol. Biol. 184, 99-105). The deduced TonB amino acid sequence contains three potential methionine start codons in the first six codons of the open reading frame. In this report, we show, by Edman degradation of [35S]methionine-labeled protein, that TonB protein synthesized in vitro initiates at the third of these methionine codons. A method for detecting TonB synthesized in vivo has been developed that involves expression of TonB from the lambda PL promoter and pulse labeling with [35S]methionine. TonB synthesized in vivo has a chemical half-life of 10 min at 42 degrees C. It is exported from the cytoplasm, as determined by proteinase K accessibility experiments. It fractionates with spheroplasts under conditions where maltose-binding protein fractionates with the periplasm. It has the same mobility in three different polyacrylamide gel systems as TonB synthesized in vitro. We concluded that the amino terminus of TonB is uncleaved following its export from the cytoplasm and that TonB is a membrane-associated protein. Characterization of a tonB-phoA gene fusion suggests that the amino-terminal 41 amino acids of TonB are sufficient to promote export of the fusion protein and presumably TonB as well. Models for TonB orientation within the cell envelope are presented.     

Mitochondrial targeting signal of rat liver monoamine oxidase B is located at its carboxy terminus.

Monoamine oxidase B, a typical intrinsic protein of the outer mitochondrial membrane, has an uncleavable targeting signal and is inserted into the membrane without proteolytic maturation. To investigate the region responsible for targeting the enzyme to the outer mitochondrial membrane, various mutated proteins were expressed in cultured mammalian cells, and the distributions of the expressed proteins were analyzed by immunofluorescence microscopy and subcellular fractionation. Deletion of the carboxy-terminal 28 amino acids of monoamine oxidase B abolished the transfer of the enzyme to mitochondria, while the deletion of the amino-terminal 55 amino acids had no effect on the transfer to mitochondria. The existence of the targeting signal at the carboxy-terminal portion of the enzyme was confirmed by using hybrid proteins in which the amino- or carboxy-terminal portion of the enzyme was fused to the hydrophilic portion of cytochrome b5. The fused protein with the carboxy-terminal 29 amino acid residues of monoamine oxidase B was localized in mitochondria, whereas that with 10 amino acids remained in the cytoplasm. These results indicate that the targeting signal of monoamine oxidase B is present within its carboxy-terminal 29 amino acid residues.     

A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein.

Cleavage of the Gag and Gag-Pol polyprotein precursors is a critical step in proliferation of retroviruses and retroelements. The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag protein. Ty1 Gag is not obviously homologous to the Gag proteins of retroviruses. The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation. Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus. A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag. Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs. While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1. Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.     

Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting.

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.     

Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent

Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.     

Structural basis for the function of Tim50 in the mitochondrial presequence translocase

Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane and the presequence translocase of the inner membrane. Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding β-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane.     

Differential expression of two functional serine/threonine protein kinases from soyabean that have an unusual acidic domain at the carboxy terminus

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::β-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses. Received: 6 January 1997 / Accepted: 19 March 1997     

Identification of the carboxy terminus as important for the isoform- specific subcellular targeting of glucose transporter proteins

Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd2+. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40 degrees C, first described by Zhang et al. (1991), is insensitive to Cd2+ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40 degrees C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type flagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of flagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different flagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell- body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.     

Antiserum specific for the carboxy terminus of the transforming protein of Rous sarcoma virus

An antiserum specific for the carboxy terminus of p60src, the transforming protein of Rous sarcoma virus, was produced by immunization of rabbits with a conjugate of bovine serum albumin and the synthetic peptide NH2-Tyr-Val-Leu-Glu-Val-Ala-Glu-COOH. The carboxy-terminal six amino acids of this peptide correspond in sequence to that deduced for the carboxy terminus of the p60src of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A. The p60src proteins of the several strains of Rous sarcoma virus and the cellular homolog of the viral transforming protein, p60c-src, comprise a polymorphic family of polypeptides. The anticarboxy-terminal serum reacted readily with the p60src proteins of three different strains of Rous sarcoma virus. In contrast, no precipitation of cellular p60c-src could be detected with this serum. This suggests that the viral p60src proteins have identical carboxy termini and that the carboxy terminus of cellular p60c-src may be different from that of viral p60src. The anticarboxy-terminal serum reacted poorly with the subpopulation of viral p60src which is present in a complex with two cellular phosphoproteins. Apparently, the presence of the two cellular proteins interferes with the recognition of p60src by the anticarboxy-terminal serum. It seems likely, therefore, that these two cellular proteins bind to the carboxy-terminal domain of p60src.     

A general method for preparation of peptides biotinylated at the carboxy terminus.

A method for the preparation of a biotinylated resin that can be elongated by standard methods of solid-phase peptide synthesis to give peptides biotinylated at the carboxy terminus is described. This methodology is particularly important for the preparation of biotinylated peptides in which a free amino terminus is required. Coupling of N epsilon-9-fluorenylmethoxycarbonyl-(Fmoc)-N alpha-tert-butyloxycarbonyl(Boc)-L- lysine to p-methylbenzhydrylamine resin, followed by removal of the Fmoc protecting group and reaction with (+)-biotin-4-nitrophenyl ester yielded N alpha-Boc-biocytin-p-methyl-benzhydrylamine resin. The utility of this resin was tested by the synthesis of a biotinylated peptide, Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-biocytin-NH2, for use as an in vitro substrate for myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation. Analysis of the peptide derivative by HPLC and mass spectrometry revealed a single major product of the expected mass, indicating that the biotin group survived cleavage and deprotection with HF. The biotinylated peptide served as a substrate for NMT, and the resulting myristoylated peptide could be quantitatively recovered by adsorption to immobilized avidin.     

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.     

Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation.