Characterization of the extra-large G protein alpha-subunit XLalphas. I. Tissue distribution and subcellular localization

Our group previously described a new type of G protein, the 78-kDa XLalphas (extra large alphas) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fractionation, XLalphas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient that contained trans-Golgi network and was differentially distributed to Galphas, which also peaked in the top fractions containing plasma membrane. Here, we investigate, using a new antibody specific for the XL domain, the tissue distribution and subcellular localization of XLalphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence analysis of various adult rat tissues, XLalphas and XLN1 were found to be enriched in neuroendocrine tissues, with a particularly high level of expression in the pituitary. By both immunofluorescence and immunogold electron microscopy, endogenous as well as transfected XLalphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immunoreactivity on internal, perinuclear membranes. Upon subcellular fractionation, immunoreactive XLalphas behaved similarly to Galphas but was differentially distributed to ADP-ribosylated XLalphas. Moreover, the bottom fractions of the velocity sucrose gradient were found to contain not only trans-Golgi network membranes but also certain subdomains of the plasma membrane, which reconciles the present with the previous observations. To further investigate the molecular basis of the association of XLalphas with the plasma membrane, chimeric proteins consisting of the XL domain or portions thereof fused to green fluorescent protein were analyzed by fluorescence and subcellular fractionation. In both neuroendocrine and non-neuroendocrine cells, a fusion protein containing the entire XL domain, in contrast to one containing only the proline-rich and cysteine-rich regions, was exclusively localized at the plasma membrane. We conclude that the physiological role of XLalphas is at the plasma membrane, where it presumably is involved in signal transduction processes characteristic of neuroendocrine cells.     

Detection of DNA fragmentation and apoptotic proteins, and quantification of uncoupling protein expression by real-time RT-PCR in adipose tissue

Better understanding of the mechanisms involved in adipose tissue growth and metabolism is critical for the development of more effective treatments for obesity. However, because of its high lipid and low protein content, adipose tissue can present unique problems in some experimental procedures. We describe three protocols that provide new or improved methods for analysis of DNA, RNA, and protein from different adipose tissues. The first protocol provides a simple and rapid method for separation of fragmented DNA and visualization of apoptotic DNA laddering without the need for radioisotopes. This technique allows for an estimate of the amount of DNA fragmentation, and hence, apoptosis. The second protocol details subcellular fractionation of adipose tissue for the extraction of protein in the mitochondrial and cytosol fractions and the measurement of apoptotic protein (Bcl-2 and Bax) levels in each fraction. The last protocol involves extraction of total RNA from adipose tissue and the measurement of uncoupling protein mRNA using real-time RT-PCR, a method that has not previously been used to measure expression of uncoupling proteins in adipose tissue.     

A framework for the automated analysis of subcellular patterns in human protein atlas images

The systematic study of subcellular location patterns is required to fully characterize the human proteome, as subcellular location provides critical context necessary for understanding a protein's function. The analysis of tens of thousands of expressed proteins for the many cell types and cellular conditions under which they may be found creates a need for automated subcellular pattern analysis. We therefore describe the application of automated methods, previously developed and validated by our laboratory on fluorescence micrographs of cultured cell lines, to analyze subcellular patterns in tissue images from the Human Protein Atlas. The Atlas currently contains images of over 3000 protein patterns in various human tissues obtained using immunohistochemistry. We chose a 16 protein subset from the Atlas that reflects the major classes of subcellular location. We then separated DNA and protein staining in the images, extracted various features from each image, and trained a support vector machine classifier to recognize the protein patterns. Our results show that our system can distinguish the patterns with 83% accuracy in 45 different tissues, and when only the most confident classifications are considered, this rises to 97%. These results are encouraging given that the tissues contain many different cell types organized in different manners, and that the Atlas images are of moderate resolution. The approach described is an important starting point for automatically assigning subcellular locations on a proteome-wide basis for collections of tissue images such as the Atlas.     

Assessment of ProteoExtract subcellular fractionation kit reveals limited and incomplete enrichment of nuclear subproteome from frozen liver and heart tissue

The nuclear fraction of the ProteoExtract subcellular fractionation kit was assessed using frozen rat liver and heart tissue. Fractionation was evaluated by Western blot using protein markers for various subcellular compartments and followed up with LC/MS/MS analysis of the nuclear fractions. Of the proteins identified, nuclear proteins were in the minority (less than 15%) and there was poor representation of the various nuclear substructures when compared with liver nuclear isolations using a classical density‐based centrifugation protocol. The ProteoExtract kit demonstrated poor specificity for the nucleus and offers limited promise for proteomics investigations of the nuclear subproteome in frozen tissue samples.     

Digestion of an exogenous protein by rat yolksac cultured in vitro

Yolk-sacs were removed from 17.5-day pregnant rats injected 2-5h previously with (125)I-labelled bovine serum albumin. The specific activities of acid phosphatase and acid proteinase, and the specific radioactivities (trichloroacetic acid-insoluble and trichloroacetic acid-soluble) were measured in subcellular fractions prepared by homogenization and differential centrifugation. The conversion of acid-insoluble into acid-soluble radioactivity within cultured tissue was followed and the nature of the liberated products was investigated by gel chromatography. The results are consistent with the protein entering lysosomes and being digested there. The radiolabel was released chiefly as free iodotyrosine.     

STUDIES ON THE RELATIONSHIP BETWEEN GABA SYNTHESIS AND PROTEIN SYNTHESIS IN BRAIN

Abstract— The synthesis of γ-aminobutyric acid (GABA) in mouse brain was decreased by treatment of the animals with pyridoxal phosphate- γ-glutamylhydrazone, an inhibitor of glutamate decarboxylase in vivo. Under these experimental conditions the following parameters were studied: (1) the incorporation of labeled leucine in vivo , into protein of brain subcellular fractions; (2) the brain polysome profile; (3) the incorporation of labeled leucine into protein in vitro , in ribosomal preparations isolated from brain tissue. In other experiments, GABA synthesis was also decreased in brain cortex slices by preincubation with aminooxyacetic acid. The incorporation of [³H]leucine or [¹⁴C]leucine into protein in these slices was studied, and samples from the proteins were subjected to acrylamide-sodium dodecylsulfate gel electrophoresis. Radioactivity was counted in slices of the gel. The results of the experiments in vivo and in vitro indicate that the previously reported decrease of protein synthesis induced by an inhibition of GABA synthesis affects proteins of all subcellular fractions and all populations of protein as separated by gel electrophoresis. The polysome profile from brains of mice with decreased GABA synthesis was similar to that of control mice. This result differs from that found when brain protein synthesis is inhibited by dopamine and serotonin.     

Chemical pathology of neurofibrils. Neurofibrillary tangles of Alzheimer's presenile-senile dementia.

A subcellular fraction enriched in twisted tubules was obtained by differential centrifugation of a homogenate of neurons isolated from areas of the brain with many neurofibrillary tangles from patients with Alzheimer's presenile-senile dementia. A unique protein (molecular weight 50,000 daltons) which does not co-migrate with either of the two tubulin monomers of the major neurofilament protein, both purified from human brain, was found in this subcellular fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similarly processed tissue from areas of the brain poor in neurofibrillary tangles contained low levels of this new protein. The new protein band could not be seen in control patients.     

Protein Kinase C Activity in Rat Brain Cortex

The procedure used to obtain cerebral tissue for analysis of protein kinase C (PKC) activity may affect the subcellular distribution of the enzyme. We compared different methods of tissue preparation and found that the proportion of PKC activity associated with the particulate fraction of the cerebral cortex was only 30% when the brain was frozen in situ while the animal was on life support or after decapitation followed by delayed freezing. Other methods of obtaining cerebral tissue resulted in 49-56% of the PKC activity in the particulate fraction. Freezing per se had no apparent effect on the activity or subcellular distribution of PKC. In addition, whenever the particulate PKC activity was high (greater than 48%), there was also a significant increase in the proportion of particulate protein (from 51 to approximately 63%, p less than 0.05).     

Reproducibility,sensitivity and compatibility of the ProteoExtract® subcellular fractionation kit with saturation labeling of laser microdissected tissues

Laser microdissection (LMD), a method of isolating specific microscopic regions of interest from a tissue that has been sectioned, is increasingly being applied to study proteomics. LMD generally requires tissues to be fixed and histologically stained, which can interfere with protein recovery and subsequent analysis. We evaluated the compatibility and reproducibility of protein extractions from laser microdissected human colon mucosa using a subcellular fractionation kit (ProteoExtract^®, Calbiochem). Four protein fractions corresponding to cytosol (fraction 1), membrane/organelle (fraction 2), nucleus (fraction 3) and cytoskeleton (fraction 4) were extracted, saturation labeled with Cy5 and 5 μg separated by both acidic (pH 4–7) and basic (pH 6–11) 2‐DE. The histological stains and fixation required for LMD did not interfere with the accurate subcellular fractionation of proteins into their predicted fraction. The combination of subcellular fractionation and saturation CyDye labeling produced very well resolved, distinct protein spot maps by 2‐DE for each of the subcellular fractions, and the total number of protein spots consistently resolved between three independent extractions for each fraction was 893, 1128, 1245 and 1577 for fractions 1, 2, 3 and 4, respectively. Although significant carryover of protein did occur between fractions, this carryover was consistent between experiments, and very low inter‐experimental variation was observed. In summary, subcellular fractionation kits are very compatible with saturation labeling DIGE of LMD tissues and provide greater coverage of proteins from very small amounts of microdissected material.     

Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy

We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.     

Protein kinase activity in rat testis interstitial tissue. Effect of luteinizing hormone and other factors.

Protein kinase activity was determined in subcellular fractions of rat testis interstitial tissue after incubation of the intact tissue with LH (luteinizing hormone) in vitro. Various factors that might have changed the activity of this enzyme during preparation of the fractions before assay were also investigated. The following results were obtained. 1. LH and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) added together during incubation of the interstitial tissue caused a twofold increase in the protein kinase activity in the total tissue homogenate and subcellular fractions (12000g X 5 min pellet and 105000g X 60 min supernatant and pellet). 2. A decrease of approx. 40% in the total amount of protein kinase recovered in the soluble fraction (105000g supernatant) occurred in tissue incubated with LH and 3-isobutyl-1-methylxanthine when compared with the controls. No change in total activity was found in the other fractions. 3. LH and 3-isobutyl-1-methylxanthine caused an increase in cyclic AMP concentration in the soluble fraction (from 30 +/- 6 to 450 +/- 40 pmol/mg of protein, means +/- S.E.M., n = 4), but there was little or no increase in the particulate fractions [from 9 +/- 1 to 13 +/- 3 pmol/mg of protein (n = 3) and from 6 +/- 2 to 23 +/- 11 pmol/mg of protein (n = 3) in the 12000g and 105000g pellets respectively]. 4 Addition of 3-isobutyl-1-methylxanthine alone had little effect on protein kinase activity or cyclic AMP concentrations. 5. Little or no protein kinase activity could be demonstrated in subcellular particulate fractions unless Triton X-100 was added; the effect of this detergent was shown to be at least partly due to the inhibition of adenosine triphosphatase activity. 6. In the presence of Triton X-100 approx. 57% of the total protein kinase activity in the homogenate was found in the 105000g supernatant compared with 11% in the 105000g pellet and 32% in the 12000g pellet. 7. In contrast with adipose-tissue protein kinase [Corbin et al. (1973) J. Biol. Chem. 248, 1813-1821] the relative amounts of cyclic AMP-dependent and -dependent enzyme were not affected by dilution of the interstitial-tissue fractions. NaCl (0.5 M) decreased the estimated total amount of protein kinase activity.     

The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation

1. On incubating cerebral-cortex slices at 37° in an oxygenated medium marked changes resulted in the subcellular distribution of proteins and phosphoproteins in the tissue. The protein content of the nuclear fraction more than doubled, whereas the yields of microsomal and supernatant proteins were both markedly decreased. The amount of phosphoprotein/mg. of protein decreased in the microsomal and supernatant fractions, but showed little change in the nuclear and mitochondrial fractions. The loss of microsomal protein could be partly prevented by rinsing the slices briefly in cold sucrose solution before dispersion; the altered subcellular distribution was apparently related to contamination of the dispersing solution with traces of salts from the medium. 2. The subcellular location of the phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro has been reinvestigated by two different procedures. Comparison between unstimulated and stimulated slices after incubation in the presence of [³²P]orthophosphate showed that phosphoprotein radioactivity increased on stimulation to a greater extent in a membrane-rich fraction than in a mitochondria-rich fraction, these being obtained by immediate density-gradient fractionation of the tissue dispersion. With fractions isolated by differential centrifuging the percentage increase in a combined mitochondrial and nuclear fraction was 5% as compared with 24% (P<0·02) in the microsomal fraction and 30% in the original dispersion before fractionation. The sensitive phosphoprotein therefore appears to be located in structures sedimenting with the microsomal fraction, rather than with the nuclear fraction as previously claimed.     

The PLAC1 protein localizes to membranous compartments in the apical region of the syncytiotrophoblast

PLAC1 is a trophoblast-specific gene that maps to a locus on the X-chromosome important to placental development. We have previously shown that PLAC1 gene expression is linked to trophoblast differentiation. The objective of this study was to define the localization of the PLAC1 polypeptide as a prerequisite to understanding its function. Polyclonal antibodies specific for the putative PLAC1 polypeptide were generated. The subcellular localization of PLAC1 in the trophoblast was examined by immunohistochemical analysis of human placenta complemented by immunoblot analysis of subcellular fractions. Brightfield immunohistochemical analysis of placental tissue indicated that the PLAC1 protein localizes to the differentiated syncytiotrophoblast in the apical region of the cell. Deconvlution immunofluorescence microscopy confirmed localization to the apical region of the syncytiotrophoblast. Its distribution included both intracellular compartments as well as loci in close association with the maternal-facing, microvillous brush border membrane (MVM). These findings were supported by immunoblot analysis of subcellular fractions. A 30 kDa band was associated with the microsomal fraction of placental lysates but not the mitochondrial, nuclear, or soluble fractions, suggesting PLAC1 is targeted to a membrane location. Plasma membranes were obtained from the fetal-facing, basal surface (BM) and the maternal-facing, MVM of the syncytiotrophoblast membrane. PLAC1 immunoreactivity was only detected in membrane fractions derived from the apical MVM consistent with immunohistochemical analyses. These data demonstrate that the PLAC1 protein is restricted primarily to the differentiated trophoblast, localizing to intracellular membranous compartment(s) in the apical region of the syncytiotrophoblast and associated with its apical, microvillous membrane surface.     

Effect of ion removal on the solubility of rat brain proteins in chloroform-methanol mixtures

—The amount of chloroform-methanol-soluble protein obtained from rat brain tissue homogenates which have been subjected to washing by repeated contrifugation or dialysis is a several-fold greater than that obtained from untreated homogenates. The increase consequence of the removal of loosely bound electrolytes during the process of centrifugation or dialysis: little or no increase is observed upon (a) the addition of the supernatant, the ashed supernatant, or the diffusate to the washed homogenate; (b) the addition of inorganic salts to the washed homogenate; or (c) washing of the homogenate with a salt solution. The previously observed effect of sucrose in increasing the amount of chloroform–methanol-soluble protein obtained from subcellular fractions is apparently superimposed upon the effect of the removal of salts.     

Studies on protein phosphorylation using subcellular fractions from insulin-treated white adipose tissue of rats

In these studies the incorporation of 32P into proteins within subcellular fractions, obtained from rat white adipose tissue upon incubation in the presence of [gamma-32P]ATP, was investigated. A stable increase in the activity of protein serine(threonine) kinase in high-speed supernatant fractions was observed following treatment of intact tissue with insulin. Protein kinase activity associated with the plasma membrane fraction of cells was diminished in response to insulin, but the decrease was apparently insufficient to account for increases observed in corresponding supernatant fractions. A range of assay conditions was employed to characterize the insulin-stimulated protein serine(threonine) kinase in in supernatant fractions. The insulin-stimulated protein serine(threonine) kinase displays properties that indicate it is distinct from a number of well-characterized protein kinases, including those regulated by cAMP, calcium ions (in the presence or absence of calmodulin or mixtures of phosphatidylserine-diacylglycerol), polyamines, or heparin. There were no apparent effects of insulin on incorporation of 32P into added casein or histones II-S or III-S. The protein serine(threonine) kinase activity (or activities) described here displays properties that also appear to differ from the properties of previously described insulin-stimulated activities able to catalyze the phosphorylation of the ribosomal protein S6. The differences in properties may, in part, be explained by the use of different cell types, but may also indicate that treatment of cells with insulin leads to activation of more than one protein serine(threonine) kinase.     

Analysis of the dorsal spinal cord synaptic architecture by combined proteome analysis and in situ hybridization

The proteomic analysis of tissue samples is an analytical challenge, because identified gene products not only have to be assigned to subcellular structures, but also to cell subpopulations. We here report a strategy of combined subcellular proteomic profiling and in situ hybridization to assign proteins to subcellular sites in subsets of cells within the dorsal region of rat spinal cord. With a focus on synaptic membranes, which represent a complex membrane protein structure composed of multiple integral membrane proteins and networks of accessory structural proteins, we also compared different two-dimensional gel electrophoresis systems for the separation of the proteins. Using MALDI mass spectrometric protein identification based on peptide mass fingerprints, we identified in total 122 different gene products within the different synaptic membrane subfractions. The tissue structure of the dorsal region of the spinal cord is complex, and different layers of neurons can be distinguished neuroanatomically. Proteomic data combined with an in situ hybridization analysis for the detection of mRNA was used to assign selected gene products, namely the optical atrophy protein OPA-1, the presynaptic cytomatrix protein KIAA0378/CAST1, and the uncharacterized coiled-coil-helix-coiled-coil-helix domain containing protein 3 (hypothetical protein FLJ20420), to cell subsets of the dorsal area of the spinal cord. Most striking, KIAA0378/CAST1 mRNA was found only sparsely within the dorsal horn of the spinal cord, but highly abundant within the dorsal root ganglion. This finding, combined with the identification of KIAA0378/CAST1 within the synaptic membrane fraction of the spinal cord at the protein level, are consistent with the reported presynaptic localization of CAST, predominantly within the tissue we investigated primarily attributable to primary afferent sensory neurons. Our approach may be of use in broader studies to characterize the proteomes of neural tissue.     

Activity and subcellular distribution of protein kinase dependent on adenosine 3':5'-monophosphate in liver from normal and adrenalectomized rats.

We have examined whether glucocorticoids control the activity and (or) the subcellular distribution of protein kinase dependent on cyclic AMP (adenosine 3':5'-monophosphate), since they influence cyclic-AMP-dependent responses to other hormones. Protein kinase activity was determined in rat liver homogenates and subcellular fractions, nuclear, large granular, microsomal and supernatant obtained by differential sedimentation in 0.25 M sucrose. 63% of the tissue protein kinase activity detected in absence of cyclic AMP reside in the particulate fractions. Upon addition of exogenous cyclic AMP, protein kinase activity is stimulated 1.8, 1.2, 1.2 and 4.5-fold in nuclear, large granular, microsomal and supernatant fractions, respectively. Under these conditions, 66% of tissue activity are found in the supernatant fraction. The activity sensitive to exogenous cyclic AMP resolves into a major (84%) cytosoluble and a minor (16%) nucleomicrosomal component. The latter activity resists elution with isotonic saline and is increased in the presence of Triton X-100. Three groups of rats were studied: control and adrenalectomized with or without cortisol treatment. In whole liver homogenates, both protein kinase activity detected in absence of exogenous cyclic AMP and sensitivity of the enzyme to cyclic AMP were comparable in all groups. Moreover, the distribution patterns of proteins kinase activity amoung the fractions were essentially the same in all groups of animals, whether or not particles had been treated with Triton X-100. Finally, in cell-free experiments, glucocorticoids alone or in combination with their intracellular receptor did not modify protein kinase activity of rat liver. Thus the results reported do not support the possibility that glucocorticoids influence cyclic AMP-dependent protein kinase in rat liver. Yet, this study provides data, not available before, on subcellular distribution of this enzyme in rat liver.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.