Peroxisomal alanine : glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana.

At least two glyoxylate aminotransferases are hypothesized to participate in the steps of photorespiration located in peroxisomes. Until recently, however, genes encoding these enzymes had not been identified. We describe the isolation and characterization of an alanine : glyoxylate aminotransferase (AGT1, formerly AGT) cDNA from Arabidopsis thaliana. Southern blot analysis confirmed that Arabidopsis AGT1 is encoded by a single gene. Homologs of this class IV aminotransferase are also known in other plants, animals, and methylotrophic bacteria, suggesting an ancient evolutionary origin of this enzyme. AGT1 transcripts were present in all tissues of Arabidopsis, but were most abundant in green, leafy tissues. Purified, recombinant Arabidopsis AGT1 expressed in Escherichia coli catalyzed three transamination reactions using the following amino donor : acceptor combinations: alanine : glyoxylate, serine : glyoxylate, and serine : pyruvate. AGT1 had the highest specific activity with the serine : glyoxylate transamination, and apparent Km measurements indicate that this is the preferred in vivo reaction. In vitro import experiments and subcellular fractionations localized AGT1 to peroxisomes. Sequence analysis of the photorespiratory sat mutants revealed a single nucleotide substitution in the AGT1 gene from these plants. This transition mutation is predicted to result in a proline-to-leucine substitution at residue 251 of AGT1. When this mutation was engineered into the recombinant AGT1 protein, enzymatic activity using all three donor : acceptor pairs was abolished. We conclude that Arabidopsis AGT1 is a peroxisomal photorespiratory enzyme that catalyzes transamination reactions with multiple substrates.

Hsp90-binding immunophilins in plants: the protein movers.

Studies of cytoplasmic-nuclear trafficking of the glucocorticoid receptor in mammalian cells suggest that the hsp90/hsp70-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in targeted movement of the receptor along microtubule tracts. Over the past few years, plant cells have been found to possess a similar multiprotein chaperone machinery. Plant cells also contain high molecular weight FKBPs that bind to plant hsp90 via a conserved protein interaction involving tetratricopeptide repeat domains. The hsp90/hsp70-based machinery and the plant FKBPs might be used to target the trafficking of signalling proteins in plants.

Peroxin puzzles and folded freight: peroxisomal protein import in review.

Peroxisomes are organelles that perform a variety of functions, including the metabolism of hydrogen peroxide and the oxidation of fatty acids. Peroxisomes do not possess organellar DNA; all peroxisomal matrix proteins are post-translationally translocated into the organelle. The mechanism of peroxisomal protein translocation has been the subject of vigorous research in the past decade. Many of the proteins (peroxins, abbreviated Pex) that play critical roles in peroxisome biogenesis have been identified through functional complementation of yeast strains and of Chinese hamster ovary cell lines that are defective in peroxisome biogenesis. Researchers are now turning towards biochemical and genetic analyses of these peroxins to define their roles in peroxisome biogenesis and to discover interacting protein partners. Evidence suggests that some of the interacting partners include molecular chaperones. Several current models for peroxisomal protein import are presented.

The surprising complexity of peroxisome biogenesis.

Peroxisomes are small organelles with a single boundary membrane. All of their matrix proteins are nuclear-encoded, synthesized on free ribosomes in the cytosol, and post-translationally transported into the organelle. This may sound familiar, but in fact, peroxisome biogenesis is proving to be surprisingly unique. First, there are several classes of plant peroxisomes, each specialized for a different metabolic function and sequestering specific matrix enzymes. Second, although the mechanisms of peroxisomal protein import are conserved between the classes, multiple pathways of protein targeting and translocation have been defined. At least two different types of targeting signals direct proteins to the peroxisome matrix. The most common peroxisomal targeting signal is a tripeptide limited to the carboxyl terminus of the protein. Some peroxisomal proteins possess an amino-terminal signal which may be cleaved after import. Each targeting signal interacts with a different cytosolic receptor; other cytosolic factors or chaperones may also form a complex with the peroxisomal protein before it docks on the membrane. Peroxisomes have the unusual capacity to import proteins that are fully folded or assembled into oligomers. Although at least 20 proteins (mostly peroxins) are required for peroxisome biogenesis, the role of only a few of these have been determined. Future efforts will be directed towards an understanding of how these proteins interact and contribute to the complex process of protein import into peroxisomes.

The effects of chaperones and the influence of protein assembly on peroxisomal protein import.

Peroxisomal proteins are synthesized in the cytoplasm and post-translationally translocated into the organelle. The role of chaperones and protein folding in peroxisomal protein transport is still unclear. Translocation of proteins into mitochondria requires that precursor proteins assume an extended conformation; cytosolic chaperones are thought to help maintain this conformation. In contrast, peroxisomal protein import does not require unfolding of the targeted protein. However, the molecular chaperones Hsp70 and Hsp40 may be important for translocation. We present several lines of evidence that show that plant peroxisomal protein import is enhanced by chaperones. First, peroxisomes isolated from heat-shocked pumpkin seedling tissues exhibited increased protein import relative to control peroxisomes. Second, antibodies raised against wheat germ cytosolic Hsp70 and Escherichia coli Hsp90 inhibited import of the peroxisomal protein isocitrate lyase. To our knowledge, this is the first time that Hsp90 has been directly implicated in a protein transport event. Third, peroxisomal proteins were immunoprecipitated by wheat germ Hsp70 antibodies. We also present results that suggest that the efficiency of peroxisomal protein import is influenced by the structure of the targeted protein; monomeric isocitrate lyase was imported more efficiently than oligomeric isocitrate lyase. Taken together, these data demonstrate that the assembly state of peroxisomal proteins and the chaperones that may mediate those states are both important for efficient peroxisomal protein import.

Nucleotide triphosphates are required for the transport of glycolate oxidase into peroxisomes.

All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

Protein transport into higher plant peroxisomes. In vitro import assay provides evidence for receptor involvement.

Peroxisome biogenesis requires that proteins be transported from their site of synthesis in the cytoplasm to their final location in the peroxisome matrix or membrane. Glyoxysomes are a class of peroxisomes found primarily in germinating seedlings and are involved in mobilizing fatty acids via the glyoxylate cycle and the beta-oxidation pathway. We have used an in vitro assay to study the mechanism(s) of import of proteins into glyoxysomes. Results from this assay indicate that the transport process is time- and temperature-dependent and is specific for peroxisomal proteins. Isocitrate lyase, a glyoxysomal protein, and the leaf-type peroxisomal enzyme glycolate oxidase (GLO) were transported into pumpkin (Cucurbita pepo) glyoxysomes with no apparent differences in efficiency of import. Thus, this in vitro assay appears to be physiologically relevant and correlates well with expected in vivo conditions. Protein import was also energy-dependent and saturable. Nonradiolabeled GLO competed with radiolabeled, in vitro-synthesized GLO for components of the import machinery. Finally, pretreatment of the isolated glyoxysomes with protease virtually abolished subsequent import of GLO. Taken together, these results indicate that a proteinaceous receptor is involved in the import of peroxisomal proteins.

Assessing risk in families affected by substance abuse.

A new instrument assessing risk in substance abuse-affected families is presented. The instrument assesses those dimensions of substance abuse that make it more difficult for parents to meet the basic needs of their children. It also assesses those components of the parent's environment that may increase risk to the family's well-being. Information is presented on the instrument's reliability and validity. The article concludes with a discussion of the issues involved in assessing the risk for child abuse and neglect in families where there is an identified substance abuse problem.

Substance abuse-affected families in the child welfare system: new challenges, new alliances.

During the past decade a record number of single-parent families entered the child welfare system because the mother had an identified problem of substance abuse. These trends have forced child welfare agencies and substance abuse treatment providers to take a new look at the needs of mothers with problems of chemical dependency and their children. This article examines the issues that face child welfare and substance abuse treatment professionals as they attempt to address these new challenges and recommends strategies for forging new alliances and closing gaps in service delivery. The article calls for action to end the oppression that hampers efforts to effectively serve these families.

Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant.

Higher plants possess several classes of peroxisomes that are present at distinct developmental stages and serve different metabolic roles. To investigate the cellular processes that regulate developmental transitions of peroxisomal function, we analyzed the targeting of glyoxysomal proteins to leaf-type and root peroxisomes. We transferred genes encoding the glyoxysome-specific enzymes isocitrate lyase (IL) and malate synthase into Arabidopsis plants and showed, in cell fractionation and immunogold localization experiments, that the glyoxysomal proteins were imported into leaf-type and root peroxisomes. We next defined the sequences that target IL to peroxisomes and asked whether the same targeting determinant is recognized by different classes of the organelle. By localizing deletion and fusion derivatives of IL, we showed that the polypeptide's carboxyl terminus is both necessary for its transport to peroxisomes and sufficient to redirect a passenger protein from the cytosol to both glyoxysomes and leaf-type peroxisomes. Thus, glyoxysomal proteins are transported into several classes of peroxisomes using a common targeting determinant, suggesting that protein import does not play a regulatory role in determining a peroxisome's function. Rather, the specific metabolic role of a peroxisome appears to be determined primarily by processes that regulate the synthesis and/or stability of its constituent proteins. These processes are specified by the differentiated state of the cells in which the organelles are found.

The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.

Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.

ATP is required for the binding of precursor proteins to chloroplasts.

One of the first steps in the transport of nuclear-encoded, cytoplasmically synthesized precursor proteins into chloroplasts is a specific binding interaction between precursor proteins and the surface of the organelle. Although protein translocation into chloroplasts requires ATP hydrolysis, binding is generally thought to be energy independent. A more detailed investigation of precursor binding to the surface of chloroplasts showed that ATP was required for efficient binding. Protein translocation is known to require relatively high levels (1 mM or more) of ATP. As little as 50-100 microM ATP caused significant stimulation of precursor binding over controls with no ATP. Several different precursors were tested and all showed increased binding upon addition of low levels of ATP. Nonhydrolyzable analogs of ATP did not substitute for ATP, indicating that ATP hydrolysis was required for binding. A protonmotive force was not involved in the energy requirement for binding. Other (hydrolyzable) nucleotides could substitute for ATP but were less effective at stimulating binding. Binding was stimulated by ATP generated inside chloroplasts even when an ATP trap was present to destroy external ATP. We conclude that internal ATP is required for stimulation of precursor binding to chloroplasts.

Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes.

The energy requirements for the import of nuclear-encoded proteins into isolated chloroplasts have been reinvestigated. We have shown that, in contrast to protein import into mitochondria, the translocation of the precursors to ferredoxin, plastocyanin (prPC) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (prSS) across all chloroplastic membranes is independent of a protonmotive force and requires only ATP. This extends previous works in which investigations were limited to prSS and demonstrates that our results are probably general to all chloroplastic protein precursors. Our results are particularly interesting for the import of prPC, since in addition to the two envelope membranes, this protein must traverse the energy-transducing thylakoid membranes en route to its proper location in the thylakoid lumen. This lack of involvement of a protonmotive force, specifically of a transmembrane electric potential, demonstrates that separate mechanisms operate during the import of proteins into chloroplasts and mitochondria. We also examined the question of whether ATP is utilized inside or outside of chloroplasts during protein import. Previous attempts to resolve this question have resulted in conflicting answers. We found, by two independent approaches, that ATP for protein import is utilized inside chloroplasts. The implications of these results on the possible mechanisms of protein import into chloroplasts are discussed.