Although tRNAs participate in the essential function of protein translation in the cytoplasm, tRNA transcription and numerous processing steps occur in the nucleus. This subcellular separation between tRNA biogenesis and function requires that tRNAs be efficiently delivered to the cytoplasm in a step termed "primary tRNA nuclear export". Surprisingly, tRNA nuclear-cytoplasmic traffic is not unidirectional, but, rather, movement is bidirectional. Cytoplasmic tRNAs are imported back to the nucleus by the "tRNA retrograde nuclear import" step which is conserved from budding yeast to vertebrate cells and has been hijacked by viruses, such as HIV, for nuclear import of the viral reverse transcription complex in human cells. Under appropriate environmental conditions cytoplasmic tRNAs that have been imported into the nucleus return to the cytoplasm via the 3rd nuclear-cytoplasmic shuttling step termed "tRNA nuclear re-export", that again is conserved from budding yeast to vertebrate cells. We describe the 3 steps of tRNA nuclear-cytoplasmic movements and their regulation. There are multiple tRNA nuclear export and import pathways. The different tRNA nuclear exporters appear to possess substrate specificity leading to the tantalizing possibility that the cellular proteome may be regulated at the level of tRNA nuclear export. Moreover, in some organisms, such as budding yeast, the pre-tRNA splicing heterotetrameric endonuclease (SEN), which removes introns from pre-tRNAs, resides on the cytoplasmic surface of the mitochondria. Therefore, we also describe the localization of the SEN complex to mitochondria and splicing of pre-tRNA on mitochondria, which occurs prior to the participation of tRNAs in protein translation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G 0 -like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

Commercially available angiotensin II AT2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available AT2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and AT2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or AT2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of AT2 receptor binding or gene expression. We conclude that none of these commercially available AT2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study AT2 receptor expression.

Abstractω-Amidase [ω-amidodicarboxylate amidohydrolase, E.C. 3.5.1.3] isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase and ester hydrolysis reactions. ω-Amidase activity toward α-ketoglutaramate and α-ketosuccinamate (the α-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria and fungi. Despite its versatility, widespread occurrence and high specific activity, the enzyme has been little studied, possibly because the assay procedure previously required a substrate (α-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing α-ketoglutaramate and an assay procedure that measures α-ketoglutarate formation from α-ketoglutaramate in a 96-well-plate format. We also describe a 96-well plate assay procedure that measures ω-amidase-catalyzed hydroxaminolysis of commercially available succinamic acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic ferric chloride that can be quantitated spectrophometrically with negligible background interference. The two assay procedures (i.e. hydrolysis of α-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying ω-amidase about ∼3,600-fold from rat liver cytosol. The ratio of α-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. ω-Amidase has recently been shown to be identical to Nit2, a putative tumor suppressor protein. It is anticipated that these new assay procedures will help characterize the function of ω-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of ω-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology.An apparent pyruvate-activated glutaminase and an apparent pyruvate-activated asparaginase were discovered more than 60 years ago [1]. Subsequently, Meister and co-workers [2][3][4] showed that both activities were actually due to a composite of two enzymes, namely a pyridoxal 5′-phosphate-dependent glutamine transaminase (Eq. 1) plus ω-amidase (Eq. 2), and a pyridoxal 5′-phosphate-dependent asparagine transaminase (Eq. 4) plus ω-amidase (Eq. 5). The net reactions are shown in Eqs 3 and 6. Transamination of glutamine and asparagine yield α-ketoglutaramate (αKGM) 1 and α-ketosuccinamate (αKSM), respectively, both of which are substrates of ω-amidase, yielding α-ketoglutarate and oxaloacetate, respectively [2]. *Corresponding author: Fax 1 914 594 4058, boris_krasnikov@nymc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process er...

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is ω-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated ω-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are α-ketoglutaramate and α-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of α-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating α-keto-γ-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess α-ketoglutaramate may be neurotoxic. Moreover, α-ketosuccinamate is unstable and is readily converted to a number of hetero aromatic compounds that may be toxic. Thus, an important role of ω-amidase is to remove potentially toxic intermediates by converting α-ketoglutaramate and α-ketosuccinamate to biologically useful α-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of ω-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor