Showing papers on "Cellular compartment published in 2007"

Network of general and specialty J protein chaperones of the yeast cytosol.

Abstract: J proteins are obligate cochaperones of Hsp70s, stimulating their ATPase activity and thus allowing them to function in multiple cellular processes. In most cellular compartments, an Hsp70 works with multiple, structurally divergent J proteins. To better understand the functional specificity of J proteins and the complexity of the Hsp70:J protein network, we undertook a comprehensive analysis of 13 J proteins of the cytosol of the yeast Saccharomyces cerevisiae. Phenotypes caused by the absence of four proteins, Sis1, Jjj1, Jjj3, and Cwc23, could not be rescued by overexpression of any other cytosolic J protein, demonstrating the distinctive nature of J proteins. In one case, that of Zuo1, the phenotypic effects of the absence of a J protein could be rescued by overexpression of only one other J protein, Jjj1, which, like Zuo1, is ribosome-associated. In contrast, the severe growth phenotype caused by the absence of the cytosol's most abundant J protein, Ydj1, was substantially rescued by expression of J domain-containing fragments of many cytosolic J proteins. We conclude that many functions of Hsp70 chaperone machineries only require stimulation of Hsp70's ATPase activity by J protein partners. However, a subset of Hsp70 functions requires specific J protein partners, likely demanding either sublocalization within the compartment or binding to specific client proteins.

Visualizing the uptake of C60 to the cytoplasm and nucleus of human monocyte-derived macrophage cells using energy-filtered transmission electron microscopy and electron tomography

Abstract: Concerns have been raised over the release of C60 nanoparticles into the environment and the potential risk to human health To address these concerns it is essential to understand the pathways by which nanoparticles enter the cell, where they migrate to, and to establish whether the particles are transformed or modified within the cell Imaging the subcellular distribution of carbon-based nanoparticles is particularly challenging It is difficult to achieve high spatial resolution with sufficient image contrast to enable the nanoparticles to be identified within the cell We have exposed human monocyte-derived macrophages (HMMs) to C60 and used energy filtered transmission electron microscopy (EFTEM) to image the distribution of C60 aggregates within intracellular compartments We demonstrate that images recorded using low-loss electrons provide a significant improvement in contrast between the cellular material and the C60 allowing a clear differentiation between C60 and unstained cellular compartments and also between ordered and disordered forms of aggregated C60 We confirm that C60 is taken up by HMMs in vitro and is sequestered at several sites within the cell These sites include the cytoplasm, lysosomes, and most significantly the cell nuclei

Pho91 Is a Vacuolar Phosphate Transporter That Regulates Phosphate and Polyphosphate Metabolism in Saccharomyces cerevisiae

Abstract: Inorganic polyphosphate (poly P) is a biopolymer that occurs in all organisms and cells and in many cellular compartments. It is involved in numerous biological phenomena and functions in cellular processes in all organisms. However, even the most fundamental aspects of poly P metabolism are largely unknown. In yeast, large amounts of poly P accumulate in the vacuole during growth. It is neither known how this poly P pool is synthesized nor how it is remobilized from the vacuole to replenish the cytosolic phosphate pool. Here, we report a systematic analysis of the yeast phosphate transporters and their function in poly P metabolism. By using poly P content as a read-out, it was possible to define novel functions of the five phosphate transporters: Pho84, Pho87, Pho89, Pho90, and Pho91, in budding yeast. Most notably, it was found that the low-affinity transporter Pho91 limits poly P accumulation in a strain lacking PHO85. This phenotype was not caused by a regulatory effect on the PHO pathway, but can be attributed to the unexpected localization of Pho91 in the vacuolar membrane. This finding is consistent with the hypothesis that Pho91 serves as a vacuolar phosphate transporter that exports phosphate from the vacuolar lumen to the cytosol.

Functional Symmetry of Endomembranes

Abstract: In higher eukaryotic cells pleiomorphic compartments composed of vacuoles, tubules and vesicles move from the endoplasmic reticulum (ER) and the plasma membrane to the cell center, operating in early biosynthetic trafficking and endocytosis, respectively. Besides transporting cargo to the Golgi apparatus and lysosomes, a major task of these compartments is to promote extensive membrane recycling. The endocytic membrane system is traditionally divided into early (sorting) endosomes, late endosomes and the endocytic recycling compartment (ERC). Recent studies on the intermediate compartment (IC) between the ER and the Golgi apparatus suggest that it also consists of peripheral ("early") and centralized ("late") structures, as well as a third component, designated here as the biosynthetic recycling compartment (BRC). We propose that the ERC and the BRC exist as long-lived "mirror compartments" at the cell center that also share the ability to expand and become mobilized during cell activation. These considerations emphasize the functional symmetry of endomembrane compartments, which provides a basis for the membrane rearrangements taking place during cell division, polarization, and differentiation.

Expression of the ATP-binding cassette membrane transporter, ABCG2, in human and rodent brain microvessel endothelial and glial cell culture systems.

Abstract: Purpose The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments.

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse.

Abstract: Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.

Models of primitive cellular life: polymerases and templates in liposomes

Abstract: Nutrient transport, polymerization and expression of genetic information in cellular compartments are hallmarks of all life today, and must have appeared at some point during the origin and early evolution of life. Because the first cellular life lacked membrane transport systems based on highly evolved proteins, they presumably depended on simpler processes of nutrient uptake. Using a system consisting of an RNA polymerase and DNA template entrapped in submicrometre-sized lipid vesicles (liposomes), we found that the liposome membrane could be made sufficiently permeable to allow access of ionized substrate molecules as large as nucleoside triphosphates (NTPs) to the enzyme. The encapsulated polymerase transcribed the template-specific base sequences of the DNA to the RNA that was synthesized. These experiments demonstrate that units of genetic information can be associated with a functional catalyst in a single compartment, and that transcription of gene-sized DNA fragments can be achieved by relying solely on passive diffusion to supply NTPs substrates.

The mitochondrion as Janus Bifrons

Abstract: The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of heme, steroids, iron-sulfur clusters, and reactive oxygen and nitrogen species. Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

Multiple translational isoforms give functional specificity to serum- and glucocorticoid-induced kinase 1.

Abstract: Serum- and glucocorticoid-induced kinase 1 is a ubiquitous kinase that regulates diverse processes such as ion transport and cell survival. We report that a single SGK1 mRNA produces isoforms with different N-termini owing to alternative translation initiation. The long isoforms, 49 and 47 kDa, are the most abundant, localize to the ER membrane, exhibit rapid turnover, their expression is decreased by ER stress, activate the epithelial sodium channel (ENaC) and translocate FoxO3a transcriptional factors from the nucleus to the cytoplasm. The short isoforms, 45 and 42 kDa, localize to the cytoplasm and nucleus, exhibit long half-life and phosphorylate glycogen synthase kinase-3beta. The data indicate that activation of Sgk1 in different cellular compartments is key to providing functional specificity to Sgk1 signaling pathways. We conclude that the distinct properties and functional specialization of Sgk1 given by the N-terminus confer versatility of function while maintaining the same core kinase domain.

Transporter-mediated uptake into cellular compartments

Abstract: Active transport across biological membranes represents a critical step in the disposition of many drugs. It is now well-established that different efflux and uptake transporters such as P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) or organic anion transporting polypeptides (OATPs) are involved in the overall disposition and efficacy of numerous compounds. These proteins are mainly expressed at physiological sites of drug absorption and elimination, thus leading to diminished absorption and/or increased transporter-facilitated excretion. Moreover, drug transporters are known to be of protective significance in blood-organ barriers. On the contrary, only little is known about the relevance of transporter function on drug levels within tissues and cellular compartments, i.e. the site of action for many substances. Moreover, the pharmacokinetic processing inside the cell is characterized by uptake, metabolism and elimination. It is gradually being recognized that active uptake and/or efflux transporters may modify target concentrations at the subcellular receptor sites which in turn may have an influence on drug effects. This review will summarize current knowledge about the impact of transporter proteins on drug availability within pharmacologically relevant cellular compartments and tissues as hepatocytes, enterocytes, different blood cell types, brain, and the heart with emphasis on the potential clinical significance of these transporters.

Regulation of calnexin sub-cellular localization modulates endoplasmic reticulum stress-induced apoptosis in MCF-7 cells.

Abstract: The endoplasmic reticulum (ER) is the cellular compartment where proteins enter the secretory pathway, undergo post-translational modifications and acquire a correct conformation. If these functions are chronically altered, specific ER stress signals are triggered to promote cell death through the intrinsic apoptotic pathway. Here, we show that tunicamycin causes significant alteration of calnexin sub-cellular distribution in MCF-7 cells. Interestingly, this correlates with the absence of both tunicamycin-induced calnexin phosphorylation as well as tunicamycin-induced cell death. Under these conditions, calnexin-associated Bap31, an ER integral membrane protein, is subjected to a caspase-8 cleavage pattern within a specific sub-compartment of the ER. These results suggest that MCF-7 resistance to ER stress-induced apoptosis is partially mediated by the expression level of calnexin which in turn controls its sub-cellular localization, and its association with Bap31. These data may delineate a resistance mechanism to the ER stress-induced intrinsic apoptotic pathway.

Computational analysis of the S. cerevisiae proteome reveals the function and cellular localization of the least and most amyloidogenic proteins.

Abstract: Protein sequences have evolved to optimize biological function that usually requires a well-defined three-dimensional struc- ture and a monomeric (or oligomeric) state. These two requirements may be in conflict as the pro- pensity for b-sheet structure, which is one of the two most common regular conformations of the polypeptide chain in folded proteins, favors also the formation of ordered aggregates of multiple copies of the same protein (fibril, i.e., polymeric state). Such b-aggregation is typical of amyloid dis- eases that include Alzheimer's, Parkinson's, and type II diabetes as well as the spongiform ence- phalopaties. Here, an analytical model previously developed for evaluating the amyloidogenic poten- tial of polypeptides is applied to the proteome of the budding yeast (Saccharomyces cerevisiae). The model is based on the physicochemical properties that are relevant for b-aggregation and requires only the protein sequence as input. It is shown that b-aggregation prone proteins in yeast are accrued in molecular transport, protein biosynthe- sis, and cell wall organization processes while they are underrepresented in ribosome biogenesis, RNA metabolism, and vitamin metabolism. Fur- thermore, b-aggregation prone proteins are much more abundant in the cell wall, endoplasmic retic- ulum, and plasma membrane than in the nucleo- lus, ribosome, and nucleus. Thus, this study indi- cates that evolution has not only prevented the selection of amyloidogenic sequences in cellular compartments characterized by a high concentra- tion of unfolded proteins but also tried to exploit the b-aggregated state for certain functions (e.g. molecular transport) and in well-confined cellular environments or organelles to protect the rest of the cell from toxic (pre-)fibrillar species. Proteins 2007;68:273-278. V C 2007 Wiley-Liss, Inc.

Membrane topology of the endoplasmic reticulum to Golgi transport factor Erv29p.

Abstract: Secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII-coated intermediates Yeast Erv29p is a transmembrane protein cycling between these compartments It is conserved across species, with one ortholog found in each genome studied, including the surf-4 protein in mammals Yeast Erv29p acts as a receptor, loading a specific subset of soluble cargo, including glycosylated alpha factor pheromone precursor and carboxypeptidase Y, into vesicles As the eukaryotic secretory pathway is highly conserved, mammalian surf-4 may perform a similar role in the transport of unknown substrates Here we report the membrane topology of yeast Erv29p, which we solved by minimally invasive cysteine accessibility scanning using thiol-specific biotinylation and fluorescent labeling methods Erv29p contains four transmembrane domains with both termini exposed to the cytosol Two luminal loops may contain a recognition site for hydrophobic export signals on soluble cargo

Targeting of the Sendai Virus C Protein to the Plasma Membrane via a Peptide-Only Membrane Anchor

Abstract: Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFLKKILKLRGRR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic alpha-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.

Accumulation of A2-E in mitochondrial membranes of cultured RPE cells

Abstract: Lipofuscin occurs in association with various blinding diseases, including ARMD. Formation of lipofuscin is considered to be initiated by the inability of the RPE lysosome to degrade constituents of phagocytosed material resulting in its intralysosomal accumulation. Thus, the deposition of abnormal retinoid adducts causing the autofluorescent properties of RPE lipofuscin originates from abnormal products of the retinoid cycle contained in phagocytosed photoreceptor outer segments. The major lipofuscin retinoid conjugate A2-E was previously shown to exert toxic effects on RPE cells by directly damaging lysosomal function and structure. However, A2-E was also proposed to severely harm extralysosomal RPE cell structures during the pathogenesis of ARMD. This would require release or leakage of A2-E from the lysosomal compartment with subsequent targeting of other cellular compartments. We therefore now investigated intralysosomal accumulation, possible biodegradation, release from the lysosomal compartment and intracellular spreading of 14C-labelled A2-E in cultured human RPE cells. We specifically loaded lysosomes of cultured human RPE cells with [14C]A2-E. A linear increase of intracellular radioactivity was observed during the 4-week loading period. Cell fractionation experiments indicated that more than 90% of loaded A2-E was specifically accumulating in the lysosomes. After loading, the fate of the radioactive label was chased over a period of an additional 4 weeks. No metabolism or secretion of A2-E to the medium was detectable. Subcellular fractionation revealed that during the chase period, about 13% were shifted from the lysosomes to mitochondrial fractions. This effect was strikingly intensified when after loading the cells with the labeled retinoid, its intralysosomal concentration was boosted by an additional load with non-labeled A2-E. Thus about 44% of the label were located in mitochondria at the end of the chase period. No significant spreading to other cell compartments was detectable. Since A2-E was suggested to act as a proapoptotic molecule via a mitochondrial pathway, we postulate that upon reaching a critical intralysosomal concentration, A2-E is released from the lysosome and then specifically targets the outer mitochondrial membrane thereby initiating apoptosis of the RPE cell. This may also apply correspondingly to other lipofuscin-associated molecules that cause leakage of the lysosomal membrane.

Developmental distribution of plasma membrane Ca2+-ATPase isoforms in chick cerebellum.

Abstract: The plasma membrane Ca(2+)-ATPase (PMCA) is highly expressed in the nervous system, but little information is available about its implication in neuronal development. We have analyzed the expression and localization of different isoforms of PMCA in membrane vesicles and sections of chick cerebellum from embryonic day 10 to hatching. We found that the relative amount of each PMCA isoform and their spatiotemporal distribution in the cerebellum are directly linked to precise cellular types during the cerebellar maturation, even in a non-neural tissue as choroid plexus. Purkinje cells contain the highest diversity of PMCA isoforms of the cerebellar cortex since the moment of its morphogenesis. From embryonic day 15, the PMCA2 was highly expressed in the whole Purkinje cell, while PMCAs 1 and 3 had a more restricted distribution in the soma and dendritic branches, and these distributions were evolving according with cell maturation. Other cellular types seem to contain a specific combination of isoforms, but with a well-defined distribution pattern at late moments of development. Thus, PMCAs 1 and 3 were located in the soma of molecular layer interneurons, and only the PMCA2 was observed in granule cells at hatching. Furthermore, PMCA isoforms are also expressed in cellular compartments characterized by a high amount of synapses, suggesting a key role of these proteins in synaptogenesis and in the maturation of neuronal electrophysiological properties.

Labeling and identification of LNCaP cell surface proteins: a pilot study.

Abstract: BACKGROUND Membrane proteins provide the interface between the cell and its environment and are responsible for cell adhesion, mobility, and intracellular signaling. Previous studies have focused on the LNCaP whole cell proteome and transcriptome but little is known about proteins at the prostate cell membrane and how they change in response to androgens. MATERIALS AND METHODS Following treatment with R1881 or vehicle, membrane proteins of the prostate cancer LNCaP cell line were tagged with biotin using EZ-link sulfo-NHS-LC-biotin. Using the tag membrane proteins were purified and separated using two-dimensional gel electrophoresis and identified using mass spectrometry. E-cadherin and low density lipoprotein receptor (LDLR) were used as positive controls and also investigated following bicalutamide treatment. Membrane localization and androgen-regulation of proteins was confirmed using sub-cellular fractionation, Western blotting and microscopy. RESULTS We have demonstrated efficient and specific protein biotinylation and purification of LNCaP plasma membrane proteins using Western analysis. E-cadherin and LDLR were regulated at the cell surface in response to R1881 and bicalutamide. Mass spectrometry identified several androgen-regulated membrane associated proteins including Prx-3 and GRP78 which are known to localize to other cellular compartments as well as the plasma membrane. We confirmed the localization of the identified proteins in LNCaP cells by co-localization with E-cadherin and immunohistochemistry of prostate tissue. CONCLUSION Cell surface biotinylation is an effective technique for identifying membrane proteins in the LNCaP prostate cancer cell line. We have demonstrated the identification of androgen-regulated membrane proteins and their validation in tissue samples. Prostate 67: 943–954, 2007. © 2007 Wiley-Liss, Inc.

Evolution of the Endoplasmic Reticulum and the Golgi Complex

Abstract: By analyzing the morpho-physiological features of the Golgi complex, its relationship with the endoplasmic reticulum in different species, and the molecular machineries involved in intracellular transport, we conclude that; (1) all eukaryotic cells have either Golgi complexes or remnants thereof; (2) all eukaryotic cells have a large minimal set of proteins that are involved in intracellular transport; and (3) several indispensable molecular machines are always present in secreting eukaryotic cells Using this information, our data about mechanisms of intra-Golgi transport and phylogenetic analysis of several molecular machines, we propose a model for the evolution of the Golgi complex and the endoplasmic reticulum

Identification of Cellular Protein Phosphatase-1 Regulators

Abstract: Protein phosphatase-1 (PP1) is a major phosphoserine/phosphothreonine phosphatase that regulates multiple physiological events in all eukaryotic cells. Action of PP1 in cells is dictated by the association of PP1 catalytic subunit with one or more regulatory subunits that define both its catalytic function and subcellular localization. This chapter describes key methods used to identify PP1-binding proteins and assess their ability to modulate PP1 functions in mammalian cells. These methods include affinity isolation of cellular PP1 complexes, analysis of direct PP1 binding, modulation of PP (protein phosphatase) activity, and testing for the presence of the newly identified PP1 complex in cells and cellular compartments. Together these techniques set the foundation for further studies that can establish the physiological significance of this PP1 complex.

Detection of sub-cellular compartment localization of a molecule using a reduced affinity enzyme complementation reporter system

Abstract: Methods and compositions for detecting the sub-cellular localization of a molecule are provided. Aspects of the invention include detecting translocation of a cell-surface receptor to a sub-cellular compartment, e.g., the endosome, using a reduced affinity enzyme complementation reporter system. Also provided are systems and kits for use in practicing embodiments of the methods.