BUSCA: An integrative web server to predict subcellular localization of proteins

Here, we present BUSCA (http://busca.biocomp.unibo.it), a novel web server that integrates different computational tools for predicting protein subcellular localization. BUSCA combines methods for identifying signal and transit peptides (DeepSig and TPpred3), GPI-anchors (PredGPI) and transmembrane domains (ENSEMBLE3.0 and BetAware) with tools for discriminating subcellular localization of both globular and membrane proteins (BaCelLo, MemLoci and SChloro). Outcomes from the different tools are processed and integrated for annotating subcellular localization of both eukaryotic and bacterial protein sequences. We benchmark BUSCA against protein targets derived from recent CAFA experiments and other specific data sets, reporting performance at the state-of-the-art. BUSCA scores better than all other evaluated methods on 2732 targets from CAFA2, with a F1 value equal to 0.49 and among the best methods when predicting targets from CAFA3. We propose BUSCA as an integrated and accurate resource for the annotation of protein subcellular localization

Subcellular localization of pituitary enzymes

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells

GAP activity, but not subcellular targeting, is required for Arabidopsis RanGAP cellular and developmental functions

The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope

Different domains cooperate to target the human ribosomal L7a protein to the nucleus and to the nucleoli.

The human ribosomal protein L7a is a component of the major ribosomal subunit. We transiently expressed in HeLa cells L7a-β-galactosidase fusion proteins and studied their subcellular localization by indirect immunofluorescence staining with anti-β-galactosidase antibodies. We have identified three distinct domains responsible for the nuclear targeting of the protein: domain I, amino acids 23-51; domain II, amino acids 52-100; domain III, amino acids 101-220, each of which contains at least one nuclear localization signal (NLS). Through subcellular localization analysis of deletion mutants of L7a-β-galactosidase chimeras, we demonstrate that domain II plays a special role because it is necessary, although not sufficient, to target the chimeric β-galactosidase to the nucleoli. In fact, we demonstrate that the nucleolar targeting process requires the presence of domain II plus an additional basic domain that can be represented by an NLS or a basic stretch of amino acids without NLS activity. Thus, when multiple NLS are present, each NLS exerts distinct functions. Domain II drives nucleolar accumulation of a reporter protein with the cooperative action of a short basic amino acid sequence, suggesting a mechanism requiring protein-protein or protein-nucleic acid interactions

A Variable Polyglutamine Repeat Affects Subcellular Localization and Regulatory Activity of a Populus ANGUSTIFOLIA Protein.

Polyglutamine (polyQ) stretches have been reported to occur in proteins across many organisms including animals, fungi and plants. Expansion of these repeats has attracted much attention due their associations with numerous human diseases including Huntington's and other neurological maladies. This suggests that the relative length of polyQ stretches is an important modulator of their function. Here, we report the identification of a Populus C-terminus binding protein (CtBP) ANGUSTIFOLIA (PtAN1) which contains a polyQ stretch whose functional relevance had not been established. Analysis of 917 resequenced Populus trichocarpa genotypes revealed three allelic variants at this locus encoding 11-, 13- and 15-glutamine residues. Transient expression assays using Populus leaf mesophyll protoplasts revealed that the 11Q variant exhibited strong nuclear localization whereas the 15Q variant was only found in the cytosol, with the 13Q variant exhibiting localization in both subcellular compartments. We assessed functional implications by evaluating expression changes of putative PtAN1 targets in response to overexpression of the three allelic variants and observed allele-specific differences in expression levels of putative targets. Our results provide evidence that variation in polyQ length modulates PtAN1 function by altering subcellular localization

A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites

cis-acting sequences and trans-acting factors in the localization of mRNA for mitochondrial ribosomal proteins

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3′-UTR. Here we show by biochemical analysis that these mRNA transcripts are associated with the cytoskeleton through the microtubule network. Cytoskeleton association is functional for their intracellular localization near the mitochondrion, and the 3′-UTR is involved in this cytoskeleton-dependent localization. To identify the minimal elements required for localization, we generated DNA constructs containing, downstream from the GFP gene, deletion mutants of mitochondrial ribosomal protein S12 3′-UTR, and expressed them in HeLa cells. RT-PCR analysis showed that the localization signals responsible for mRNA localization are located in the first 154 nucleotides. RNA pulldown assays, mass spectrometry, and RNP immunoprecipitation assay experiments, demonstrated that mitochondrial ribosomal protein S12 3′-UTR interacts specifically with TRAP1 (tumor necrosis factor receptor-associated protein1), hnRNPM4 (heterogeneous nuclear ribonucleoprotein M4), Hsp70 and Hsp60 (heat shock proteins 70 and 60), and α-tubulin in vitro and in vivo