The NBDs that wouldn't die: A cautionary tale of the use of isolated nucleotide binding domains of ABC transporters

COMATOSE (CTS), the plant homologue of Adrenoleukodystrophy protein, is a full length ABC transporter localised in peroxisomes. In a recent article, we reported that the two nucleotide binding domains of CTS are not functionally equivalent in vivo. Mutations in conserved residues in the Walker A (K487A) and B (D606N) motifs of NBD1 resulted in a null phenotype, whereas identical mutations in the equivalent residues in NBD2 (K1136A and D1276N) had no detectable effect.1 In order to study the effect of these mutations on the ATPase activity of the nucleotide binding domains, we cloned and expressed the isolated NBDs as maltose binding protein (MBP) fusion proteins. We show that ATPase activity is associated with the isolated MBP-NBDs. However, mutations of amino acids located in conserved motifs did not result in striking reduction in activity despite well characterized roles in ATP binding and hydrolysis. We urge caution in the interpretation of results obtained from the study of isolated NBD fusions and their extrapolation to the mechanism of ATP hydrolysis in ABC transporter proteins

Trafficking routes to the plant vacuole: connecting alternative and classical pathways

Due to the numerous roles plant vacuoles play in cell homeostasis, detoxification, and protein storage, the trafficking pathways to this organelle have been extensively studied. Recent evidence, however, suggests that our vision of transport to the vacuole is not as simple as previously imagined. Alternative routes have been identified and are being characterized. Intricate interconnections between routes seem to occur in various cases, complicating the interpretation of data. In this review, we aim to summarize the published evidence and link the emerging data with previous findings. We discuss the current state of information on alternative and classical trafficking routes to the plant vacuole

The NBDs that wouldn't die. A cautionary tale in the use of isolated nucleotide binding domains of ABC transporters

COMATOSE (CTS), the plant homologue of Adrenoleukodystrophy protein, is a full length ABC transporter localised in peroxisomes. In a recent article, we reported that the two nucleotide binding domains of CTS are not functionally equivalent in vivo. Mutations in conserved residues in the Walker A (K487A) and B (D606N) motifs of NBD1 resulted in a null phenotype, whereas identical mutations in the equivalent residues in NBD2 (K1136A and D1276N) had no detectable effect.1 In order to study the effect of these mutations on the ATPase activity of the nucleotide binding domains, we cloned and expressed the isolated NBDs as maltose binding protein (MBP) fusion proteins. We show that ATPase activity is associated with the isolated MBP-NBDs. However, mutations of amino acids located in conserved motifs did not result in striking reduction in activity despite well characterized roles in ATP binding and hydrolysis. We urge caution in the interpretation of results obtained from the study of isolated NBD fusions and their extrapolation to the mechanism of ATP hydrolysis in ABC transporter proteins

Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells

Functional Expression of Human Adenine Nucleotide Translocase 4 in Saccharomyces Cerevisiae

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats

Molecular interactions of the mitochondrial Tim12 translocase subunit

The small Tims are chaperones that facilitate insertion of hydrophobic precursors into the inner mitochondrial membrane. We purified Tim12 and found it forms dimers that bind to Tim9. In this interaction, Tim12 undergoes structural changes that may be important for transport of its substrates in the mitochondrial carrier import pathway

Mechanisms and concepts paving the way towards a complete transport cycle of plant vacuolar sorting receptors.

Delivery of proteins to the lytic vacuole in plants is a complex cascade of selective interactions that specifically excludes residents of the endoplasmic reticulum and secreted proteins. Vacuolar transport must be highly efficient to avoid mistargeting of hydrolytic enzymes to locations where they could be harmful. While plant vacuolar sorting signals have been well described for two decades, it is only during the last 5 years that a critical mass of data was gathered that begins to reveal how vacuolar sorting receptors (VSRs) may complete a full transport cycle. Yet, the field is far from reaching a consensus regarding the organelles that could be involved in vacuolar sorting, their potential biogenesis, and the ultimate recycling of membranes and protein machinery that maintain this pathway. This review will highlight the important landmarks in our understanding of VSR function and compare recent transport models that have been proposed so that an emerging picture of plant vacuolar sorting mechanisms can be drawn

Translocation of mitochondrial inner-membrane proteins: conformation matters

Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating ‘assembly on demand’ of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process

Routes to and from the plasma membrane:Bulk flow versus signal mediated endocytosis

Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner