The Synthesis and Possible Transport of Specific Proteins by Cells Associated with Brain Capillaries

Abstract: Vinblastine causes alterations in the subcellular distribution of certain proteins synthesized by telencephalon slices. Proteins in various subcellular fractions were separated according to their molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactive proteins were determined by autofluorography. A microvascular fraction contained very high amounts of radioactivity in proteins with a molecular weight of 71,000. At least one of these proteins accumulated in the microvascular fraction when the telencephalon slices were incubated in vinblastine. At the same time these proteins became depleted in a myelinated axon fraction, microsomal fraction, and soluble/cytosol fraction. Vinblastine also affected the subcellular distribution of some proteins with a molecular weight below 27,000, but unlike the proteins of mol. wt. 71,000 none of these were synthesized at very high rates. Vinblastine did not effect the synthesis of protein in telencephalon slices, nor did it alter the subcellular fractionation of particles and organelles from slices. It is suggested that a non-neuronal vinblastine-sensitive protein translocation system is functioning within the cells of the microvascular network in telencephalon slices, and that at least one protein of 71,000 molecular weight and one protein with a molecular weight below 27,000 are transported on this system.     

Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation

Chlamydia trachomatis manipulates host cellular pathways to ensure its proliferation and survival. Translocation of host materials into the pathogenic vacuole (termed ‘inclusion’) may facilitate nutrient acquisition and various organelles have been observed within the inclusion, including lipid droplets, peroxisomes, multivesicular body components, and membranes of the endoplasmic reticulum (ER). However, few of these processes have been documented in living cells. Here, we survey the localization of a broad panel of subcellular elements and find ER, mitochondria, and inclusion membranes within the inclusion lumen of fixed cells. However, we see little evidence of intraluminal localization of these organelles in live inclusions. Using time-lapse video microscopy we document ER marker translocation into the inclusion lumen during chemical fixation. These intra-inclusion ER elements resist a variety of post-fixation manipulations and are detectable via immunofluorescence microscopy. We speculate that the localization of a subset of organelles may be exaggerated during fixation. Finally, we find similar structures within the pathogenic vacuole of Coxiella burnetti infected cells, suggesting that fixation-induced translocation of cellular materials may occur into the vacuole of a range of intracellular pathogens.     

Recent progress in predicting protein sub-subcellular locations

In the last two decades, the number of the known protein sequences increased very rapidly. However, a knowledge of protein function only exists for a small portion of these sequences. Since the experimental approaches for determining protein functions are costly and time consuming, in silico methods have been introduced to bridge the gap between knowledge of protein sequences and their functions. Knowing the subcellular location of a protein is considered to be a critical step in understanding its biological functions. Many efforts have been undertaken to predict the protein subcellular locations in silico. With the accumulation of available data, the substructures of some subcellular organelles, such as the cell nucleus, mitochondria and chloroplasts, have been taken into consideration by several studies in recent years. These studies create a new research topic, namely 'protein sub-subcellular location prediction', which goes one level deeper than classic protein subcellular location prediction.     

Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique

Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.     

Recent progress in predicting protein sub-subcellular locations

In the last two decades, the number of the known protein sequences increased very rapidly. However, a knowledge of protein function only exists for a small portion of these sequences. Since the experimental approaches for determining protein functions are costly and time consuming, in silico methods have been introduced to bridge the gap between knowledge of protein sequences and their functions. Knowing the subcellular location of a protein is considered to be a critical step in understanding its biological functions. Many efforts have been undertaken to predict the protein subcellular locations in silico. With the accumulation of available data, the substructures of some subcellular organelles, such as the cell nucleus, mitochondria and chloroplasts, have been taken into consideration by several studies in recent years. These studies create a new research topic, namely ‘protein sub-subcellular location prediction’, which goes one level deeper than classic protein subcellular location prediction.     

An approach to the isolation of biological particles using sedimentation analysis

A systematic, general approach for the design of an initial purification procedure for any biological particle is described in this communication. A series of centrifugations in fixed angle rotors has been used to obtain information on the sedimentation behavior of particles of interest (Anderson, N.G. (1967) Anal. Biochem. 23, 72-83). Refinements of this technique have facilitated the determination of sedimentation profiles of subcellular organelle markers in suspensions of murine spleen and brain. The degree of homogeneity of several particles with respect to size can be ascertained from the sedimentation profiles. Alterations in these profiles after mechanical disruption and treatment with detergents are readily measurable and have been found to be useful in both the characterization and isolation of subcellular particles. Because fixed angle rotors are used in these studies, the data obtained can be directly applied to the development of a preparatory scheme for purification of a desired particle. These methods for sedimentation analysis are readily applicable to subcellular organelles, macromolecular complexes, viruses, viral-like agents, and a variety of macromolecules.     

Protein translocation machineries: how organelles bring in matrix proteins

Eukaryotic cells contain several thousands of proteins that have to be accurately partitioned over the components of the cytoplasm (cytosol or any of the known organelles) to allow proper cell function. To this end, various specific topogenic signals have been designed as well as highly selective protein translocation machineries that ensure that each newly synthesized polypeptide reaches its correct subcellular destination or, in case of secretory proteins, is exported to the cell exterior. This contribution gives an overview regarding the principles of the main examples of polypeptide sorting and translocation, with emphasis on the function of cofactor binding in peroxisomal matrix protein import.     

Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium

Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.     

Acid hydrolases in the suckling rat small intestine I. Fractionation of mucosal homogenates

Synopsis Small intestine mucosal homogenates of suckling rats have been fractionated by centrifugation and analyzed for acid hydrolases and for biochemical markers of subcellular organelles. The results indicate that the acid hydrolases are associated with particles having sedimentation properties similar to those of mitochrondria. The acid hydrolases exhibited latent activity. Subfractionation on a continuous density gradient of sucrose in deuterium oxide demonstrated that these enzymes are associated with particles distinct from other subcellular organelles. Electron micrographs of the acid hydrolase-rich region of the gradient show the presence of numerous small electron dense bodies bounded by a unit membrane.     

Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.     

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.     

Phagosome proteomes open the way to a better understanding of phagosome function

Phagocytic cells take up microbes and other particles into membrane-bounded organelles called phagosomes. Studies on the protein and lipid composition of model phagosomes containing latex beads are the first step in a systems-biology approach to understanding how these organelles function.     

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.     

Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.     

Biotin-streptavidin-labeled oligonucleotides as probes of helicase mechanisms

Helicases use the energy from ATP hydrolysis to catalyze formation of single-stranded nucleic acids by unwinding double-stranded nucleic acids. The ATP-dependent reaction can be broken down into at least two steps: melting of the duplex and translocation of the enzyme along the nucleic acid lattice. Each step presents difficulties for study because clear end points for the reactions are not always available. For example, translocation involves the movement of the enzyme from one point along the lattice to a new position, with no net change in chemical structure of the nucleic acid. Hence, new assays have been developed in which the nucleic acid is modified to contain a "protein block" that impedes translocation of the enzyme. To prepare such protein blocks, biotin-streptavidin has been used due to the ease with which the biotin can be incorporated into nucleic acids by chemical synthesis. Several applications of oligonucleotides labeled with biotin-streptavidin for the study of helicase mechanisms are described.     

Actin associated with membranes of monoamine storage organelles.

A histo-immunofluorescence technique using anti-actin antibodies has been applied to various subcellular fractions of bovine adrenal medulla and rabbit blood platelets. In the adrenal medulla only the membranes of the chromaffin granules, but not the fractions containing other subcellular particles (microsomes, mitochondria) showed marked immunofluorescence was present in the membranes of the 5-hydroxy-tryptamine organelles as well as in other subcellular particles. It is concluded that in the adrenal medulla, actin is specifically associated with the membranes of the amine storage organelles, whereas in platelets the protein shows a rather general subcellular distribution.     

Mapping the subcellular protein distribution in three human cell lines

The subcellular locations of proteins are closely related to their function and constitute an essential aspect for understanding the complex machinery of living cells. A systematic effort has been initiated to map the protein distribution in three functionally different cell lines with the aim to provide a subcellular localization index for at least one representative protein from all human protein-encoding genes. Here, we present the results of more than 3500 proteins mapped to 16 subcellular compartments. The results indicate a ubiquitous protein expression with a majority of the proteins found in all three cell lines and a large portion localized to two or more compartments. The inter-relationships between the subcellular compartments are visualized in a protein-compartment network based on all detected proteins. Hierarchical clustering was performed to determine how closely related the organelles are in terms of protein constituents and compare the proteins detected in each cell type. Our results show distinct organelle proteomes, well conserved across the cell types, and demonstrate that biochemically similar organelles are grouped together.     

Demonstration of an insulin-insensitive storage pool of glucose transporters in rat hepatocytes and HepG2 cells.

The subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose-sensitive cytochalasin B binding and by protein immunoblotting using isoform-specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5'-nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT-2 occurred in a low-density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT-2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT-1 and GLUT-2, the two isoforms showed distinct subcellular distribution patterns: GLUT-2 was highly concentrated in LDM while very little GLUT-1 was found in this fraction, indicating that a large portion of GLUT-2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT-2, but lack the insulin-responsive glucose transporter translocation mechanism.