Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

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.     

Subcellular Localization of Simian Virus 40 Large Tumor Antigen

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.     

Biochemical Characterization of Intracellular Membranes Bearing Trk Neurotrophin Receptors

Neurotrophin receptor trafficking plays an important role in directing cellular communication in developing as well as mature neurons. However, little is known about the requirements for intracellular localization of the neurotrophin receptors in neurons. To isolate the subcellular membrane compartments containing the Trk neurotrophin receptor, we performed biochemical subcellular fractionation experiments using primary cortical neurons and rat PC12 pheochromocytoma cells. By differential centrifugation and density gradient centrifugation, we have isolated Trk-bearing compartments, suggesting distinct membranous localization of Trk receptors. A number of Trk-interacting proteins, such as GIPC and dynein light chain Tctex-1 were found in these fractions. Additionally, membranes enriched in phosphorylated activated forms of Trk receptors were found upon ligand treatment in primary neurons and PC12 cells. Interestingly, density gradient centrifugation experiments showed that Trk receptors from PC12 cells are present in heavy membrane fractions, while Trk from primary neurons are fractionated in lighter membrane fractions. These results suggest that the intracellular membrane localization of Trk can differ according to cell type. Taken together, these biochemical approaches allowed separation of distinct Trk-bearing membrane pools, which may be involved in different functions of neurotrophin receptor signaling and trafficking.     

Trypanosoma cruzi: fusogenic ability of membranes from cultured epimastigotes in interaction with human syncytiotrophoblast

Trypanosoma cruzi epimastigotes cultured in vitro were disrupted by successive freezing and thawing and subsequent sonication. The total homogenate was fractionated by differential centrifugation to obtain an enriched plasma membrane fraction. The proteins of subcellular parasite fractions were labeled with 131I and their binding to membrane fractions from human placenta syncytiotrophoblast was studied. Syncytiotrophoblast fractions enriched in plasma showed higher specific activity for binding an enriched T. cruzi plasma membrane fraction compared with other fractions of syncytiotrophoblast. The properties of this interaction were studied with digestive enzymes (trypsin and phospholipase A2). The results showed that both proteins and lipids could be involved in this interaction. The Ca2+ requirements for the membrane-membrane interaction are different for the two membranes studied. Also the enriched plasma membrane T. cruzi fraction had a higher capacity to induce fusion processes than the other subcellular fractions. The above results indicate that a preferential syncytiotrophoblast-T. cruzi interaction may occur between the two cell surfaces as compared to intracellular membranes and that the parasite surface is able to induce an instability process leading to membrane fusion. These results may have implications in regard to the mechanism of entry of the parasite into cells.     

Subcellular distribution and immunocytochemical localization of Na,K-ATPase subunit isoforms in human skeletal muscle

Summary

The expression of Na,K-ATPase isoforms was investigated in human skeletal muscle membranes isolated by subcellular fractionation. The α1, α2, α3 and β1 subunits were detectable in membranes prepared from the human soleus muscle. The α1 subunit was largely detected in a fraction enriched with plasma membranes (PM), its abundance in an Intracellular membrane fraction (IM) accounted for only 4% of that in the PM fraction. No α1 subunits were detected in membranes of sarcoplasmic reticulum (SR) origin. The PM and IM fractions were enriched with α2 subunits which were less abundant in the SR-enriched fraction. The abundance of α2 molecules within the IM fraction was about 75% of that in the PM fraction when the total protein content for the two fractions was taken into account. Immuno-cytochemical studies confirmed the localization of the α1 subunit to the muscle cell surface. The α2 subunit was also found to be present in the cell surface but the observation that α2 immuno-fluorescence was diffusely dispersed throughout the muscle fibre indicated that it was also present intracellularly, consistent with its biochemical localization in the PM and IM membrane fractions. The α3 subunit was detected largely in the PM fraction but the lack of good antibodies to this isoform precluded an analysis of its immunocytochemical localization. The β1 subunit was enriched in the PM fraction but was also detected to a modest extent in the IM. A polyclonal anti-β2 antibody, which reacted positively with both human brain microsomes and rat skeletal muscle membranes, revealed that human skeletal muscles contained no immunoreactive β2 subunits. Our results indicate that the human soleus expresses the α1 and α2 (and possibly the α3) subunits of the Na,K-ATPase and that the activity of these isoforms must be supported by the β1 subunit in this muscle.     

Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells.

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.     

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above.It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells.

The Ca2+-transport activity and intracellular localization of the translation product of cDNA for mung bean Ca2+/H+ antiporter (VCAX1) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca2+-ATPase and the antiporter, VCAX1 complemented the active Ca2+ transporters, and the microsomal membranes from the transformant showed high activity of the Ca2+/H+ antiporter. Treatment of the vacuolar membranes with a cross-linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p. The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose-density-gradient centrifugation of the microsomal fraction from mung bean. The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme. A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells. The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles. The subcellular fractionation of transformed tobacco cells confirmed the vacuolar membrane localization of the fusion protein. These results confirm that VCAX1p functions in the vacuolar membrane as a Ca2+/H+ antiporter and also suggest that VCAX1p may exist in the Golgi apparatus.     

Subcellular localization of nuclease in barley aleurone

The intracellular localization of an endonuclease (nuclease I) in barley aleurone responding to gibberellic acid was investigated by subcellular fractionation and immunocytochemistry with monoclonal and polyclonal antibodies. Organelle separations were performed with aleurone layers and protoplasts; immunefixations were carried out on protoplasts only. Nuclease was detected in fractions from isopycnic sucrose density gradients which were enriched in either endoplasmic reticulum or Golgi apparatus membranes. These two organelles were also labelled by the indirect immunogold method on thin sections. Intensive labelling of protein and developing vacuoles was observed. Therefore, as noted in other plants nuclease in barley is essentially a vacuolar enzyme.     

Comparative studies on membranes from bovine choroid plexus and rat kidney cortex.

Comparative subcellular fractionation studies on rat kidney and bovine choroid plexus using differential centrifugation and free flow electropheresis were undertaken because of the morphological and functional similarities of the epithelial cells of both tissues. The activities of three enzymes commonly used as markers for brush border membranes in kidney were measured in fractions of each tissue. γ-Glutamyl transpeptidase, alkaline phosphatase, and 5'-nucleotidase copurified in membrane fractions of renal cortex collected by differential centrifugation. Application of a similar fractionation procedure to choroid plexus gave relatively similar results, except for alkaline phosphatase, the yield of which was substantially reduced in a fraction enriched with two marker enzymes. Further fractionation of γ-glutamyl transpeptidase and alkaline phosphatase activities in these membrane fractions was achieved using free flow electropheresis. The two enzymes from kidney exhibited discrete peaks with a small separation, while the electropheretic pattern of γ-glutamyl transpeptidase from choroid plexus was biphasic. Alkaline phosphatase was observed to migrate with the more basic γ-glutamyl transpeptidase peak.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Prostaglandins E₁ and E₂ are specifically bound by particulate fractions from bovine adrenal medulla. The subcellular localization of these binding sites has been investigated by comparing their distribution in subcellular fractions obtained by differential and gradient centrifugation to those of marker enzymes for various organelles. Prostaglandin E₂ binding sites were purified about 16-fold with respect to the homogenate in a fraction which was highly enriched in plasma membranes on the basis of the activities of the marker enzymes acetylcholinesterase and calcium-dependent ATPase, which were both purified by about 12-fold in this fraction. The plasma membrane fraction contained relatively low activities of marker enzymes for mitochondria (monoamine oxidase), lysosomes (acid phosphatase), endoplasmic reticulum (glucose-6-phosphatase), Golgi (galactosyl transferase) and chromaffin granule membranes (dopamine β-hydroxylase). The only other fractions enriched in prostaglandin E₂ binding sites were those for the endoplasmic reticulum and the Golgi, in which the binding sites were purified about 4-fold and 7-fold, respectively. This is probably due mainly to contamination with plasma membranes, since calcium-dependent ATPase and acetylcholinesterase were each purified to a similar extent in these two fractions. These data suggest that the high-affinity prostaglandin E₂ binding sites of the adrenal medulla are localized primarily on the plasma membranes of the medullary cells.     

The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity

Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.     

A presumed human nuclear autoantigen that translocates to plasma membrane blebs during apoptosis

The structure and subcellular localization of a number of molecules change during apoptosis. These molecules are recognized by the immune system, leading to the development of autoimmunity when apoptotic cells fail to be effectively cleared by phagocytosis. We searched for such molecules by analyzing sera from 12 individuals who suffered from autoimmune diseases and from 3 patients with amyotrophic lateral sclerosis. One serum sample, designated 681, detected an antigen that fulfilled the above criteria. In Western blotting of lysates of human Jurkat T cells, the 681 antigen appeared as a distinct signal with a molecular mass of 60 kDa in normal cells, and 2 additional signals with faster mobilities were detected in apoptotic cells. The results of subcellular fractionation and immunofluorescence experiments revealed this antigen to be strictly localized in the nucleus of normal cells, but to be translocated to a region near the plasma membrane, to membrane blebs in particular, after the induction of apoptosis. Under conditions in which membrane blebbing was inhibited in apoptotic cells, the antigen still moved away from the nucleus, but its accumulation at the periplasmic region was completely abolished. The apparent partial cleavage and intracellular redistribution of the 681 antigen in apoptotic cells mimics changes previously reported for the nuclear autoantigen La, but the 681 antigen was clearly distinct from La. These results suggest that cleavage-dependent exit from the nucleus during apoptosis is a phenomenon common to nuclear autoantigens.     

Expression of monoclonal-antibody-defined antigens in fractions isolated from human breast carcinomas and patients' serum

The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these smaples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.     

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.     

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.     

Subcellular Localization of 5''-Nucleotidase in Rat Brain

The subcellular distribution of the ectoenzyme, 5'-nucleotidase, in cerebral cortex and cerebellum of the rat was studied both biochemically and cytochemically. The fractions were characterized biochemically by marker enzymes. The localization of 5'-nucleotidase activity was also investigated cytochemically in the myelin, synaptosomal, mitochondrial, and microsomal fractions. Biochemically 5'-nucleotidase was found to be enriched in the membrane-containing fractions, i.e., myelin, synaptosomal, and microsomal fractions. Cytochemistry showed the reaction product in the myelin fraction to be associated with myelin profiles. In the synaptosomal fraction reaction product could occasionally be seen at synaptosomal membranes, although it could not be attributed unequivocally to the synaptosome itself, since in positions with reaction product unidentifiable membrane structures could always be seen attached. Mitochondria were virtually without any reaction product. In the microsomal fraction 5'-nucleotidase activity was associated with unidentifiable membrane structures. It is concluded that 5'-nucleotidase is associated with myelin profiles and that the high activity found in the synaptosomal fraction is probably not associated with nerve ending plasma membranes.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.