Leonard H. Rome

Two species of lysosomal organelles in cultured human fibroblasts

Cultured diploid human skin fibroblasts were fractionated by a procedure that maximizes recovery of particles containing acid hydrolases. The cells were detached by controlled trypsinization, disrupted by N2 cavitation at low pressure and fractionated at 18,000 x g on a self-generating gradient of colloidal silica. This procedure separated two species of particles that could be consisered lysosomal. The denser one (peak density 1.11) was apparently free of other contaminants, but the more buoyant one (peak density 1.085) sedimented with or close to the peaks of other organelles, including mitochondria, Golgi, endoplasmic reticulum and plasma membranes. The two populations of particles contained acid hydrolases (phosphatase, six glycosidases and four cathepsins) in roughly equal proportions, displayed latency, had similar turnover of 35S-mucopolysaccharide in normal as well as in iduronidase-deficient cells, and were recipients of alpha-L-iduronidase, previously shown to be acquired by receptor-mediated endocytosis. Acid phosphatase staining of the intact fibroblasts showed residual bodies scattered throughout the cytoplasm and, near the nucleus, a prominent network of tubules and associated dilatations and knob-like enlargements. In both thin and thick sections, these appeared continuous, as if forming a three-dimensional network similar to the network described by Novikoff (1976) as GERL. Ultrastructural studies of the isolated fractions showed the denser lysosomal peak to be composed of small round or oblong acid phosphatase-positive bodies. The more buoyant peak contained the nonlysosomal organelles predicted from the biochemical markers, small acid phosphatase-positive bodies and large multivesiculated structures in which acid phosphatase was localized in a matrix surrounding apparently empty vesicles. These large structures may represent fragments of GERL. We suggest that the dense and buoyant lysosomal organelles originate primarily from residual bodies and the GERL network, respectively.

Vaults Are Up-regulated in Multidrug-resistant Cancer Cell Lines

Vaults are 13-MDa ribonucleoprotein particles composed largely of a 104-kDa protein, termed major vault protein or MVP, and a small vault RNA, vRNA. While MVP levels have been found to increase up to 15-fold in non-P-glycoprotein multidrug-resistant cell lines, the levels of vault particles have not been investigated. As both the function of vault particles and the mechanism of drug resistance in non-P-glycoprotein cells are unknown, we decided to determine whether vault synthesis was coupled to MDR. By cloning the human gene for vRNA and careful quantitation of the MVP and vRNA levels in MDR cells, we find that vRNA is in considerable excess to MVP. Sedimentation measurements of vault particles in multidrug resistance cells have indeed revealed up to a 15-fold increase in vault synthesis, coupled with a comparable shift of associated vRNA, demonstrating that vault formation is limited by expression of MVP or the minor vault proteins. The observation that vault synthesis is linked directly to multidrug resistance supports a direct role for vaults in drug resistance.

Inhibition of Prostaglandin Biosynthesis

Little evidence exists for stored prostaglandins in tissues (Jouvenaz, Nugteren, Beerthuis and Van Dorp, 1970) and a capacity of a tissue to release prostaglandins seems to reflect principally the capacity for biosynthesis of these compounds. Since prostaglandins represent a diverse family of oxygenated derivatives formed from certain polyunsaturated fatty acids, the Open image in new window Fig. 3.1 Formation and degradation of prostaglandins. biosynthesis of prostaglandins consists of many steps that involve a variety of different enzymes and cofactors. Inhibition of prostaglandin synthesis may thus occur by blocking any of the several processes that are involved. A brief summary of some of the metabolic events leading to the different prostaglandins is given in Figure 3.1. Because of the diversity of these processes and the stimuli and mediators that appear to modify these reactions it is expected that a wide variety of chemical agents will diminish the formation of prostaglandins in vivo. We will indicate some of the events of prostaglandin biosynthesis known to be influenced by chemical agents, and will suggest a logical framework for comparing the relative inhibitory activity of these different agents.

Vaults and Telomerase Share a Common Subunit, TEP1

Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly.

Unlocking vaults: organelles in search of a function

Abstract Vaults are cytoplasmic ribonucleoprotein structures that are highly conserved among diverse eukaryotes, and thousands are present per cell. This review summarizes our knowledge of vault structure and distribution, as well as our current speculations about the possible functions of vaults.

Structure of the vault, a ubiquitous celular component

BACKGROUND The vault is a ubiquitous and highly conserved ribonucleoprotein particle of approximately 13 MDa. This particle has been shown to be upregulated in certain multidrug-resistant cancer cell lines and to share a protein component with the telomerase complex. Determination of the structure of the vault was undertaken to provide a first step towards understanding the role of this cellular component in normal metabolism and perhaps to shed some light on its role in mediating drug resistance. RESULTS Over 1300 particle images were combined to calculate an approximately 31 A resolution structure of the vault. Rotational power spectra did not yield a clear symmetry peak, either because of the thin, smooth walls or inherent flexibility of the vault. Although cyclic eightfold (C8) symmetry was imposed, the resulting reconstruction may be partially cylindrically averaged about the eightfold axis. Our results reveal the vault to be a hollow, barrel-like structure with two protruding caps and an invaginated waist. CONCLUSIONS Although the normal cellular function of the vault is as yet undetermined, the structure of the vault is consistent with either a role in subcellular transport, as previously suggested, or in sequestering macromolecular assemblies.

Assembly of Vault-like Particles in Insect Cells Expressing Only the Major Vault Protein

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes ∼75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault functionin vivo.

Vault immunofluorescence in the brain: new insights regarding the origin of microglia

The developmental appearance of ameboid and ramified microglia in the rat brain has been examined by immunofluorescent localization of vaults, recently described ribonucleoprotein particles (Kedersha and Rome, 1986a). Vaults are distinct, multiarched structures of unknown function expressed by higher and lower eukaryotic species. Although vaults have been detected in all mammalian cells examined to date, they are highly enriched in macrophages. In the brain, vault antisera is highly specific for both ameboid and ramified microglia. The developmental profile of vault immunoreactivity in rat brain slices suggests that microglia enter the brain at 2 locations, with different time scales for each. The first migration, which begins before embryonic day 15 and subsides between postnatal days 7 and 14, was identified by vault immunoreactivity and Bandeiraea simplicifolia B4- isolectin (a microglia marker) staining. The cells appear to enter from blood vessels and display a ramified morphology as soon as they are detected in the brain. The second microglial migration occurs in the first postnatal week, when ameboid microglia appear in the corpus callosum and other large fiber tracts. Ameboid microglia appear to differentiate into ramified microglia between postnatal days 4 and 14. Vault immunoreactivity, as a very early microglial marker, provides new insight regarding the much-debated origin of the ramified microglia. It is quite clear that ameboid cells are not the sole source of ramified microglia because ramified cells can be detected before the influx of ameboid microglia. Colocalization studies with monocyte/macrophage markers ED1 and OX42 demonstrate that both ramified and ameboid microglia originate from monocyte lineage.

Up-regulation of vaults may be necessary but not sufficient for multidrug resistance.

Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w. vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA). Increased levels of MVP, vault‐associated vRNA and vaults have been linked directly to non‐P‐glycoprotein–mediated multidrug resistance (MDR). To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines. Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug‐sensitive cells. Furthermore, protein analysis of subcellular fractions of the drug‐sensitive, MVP‐transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line. Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines. Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up‐regulation alone is not sufficient to confer the drug‐resistant phenotype. This implies a requirement of an additional factor(s) for vault‐mediated MDR.

Vaults are the answer, what is the question?

Vaults are large cytoplasmic ribonucleoprotein (RNP) particles of eukaryotic cells, whose considerable abundance and striking evolutionary conservation argue for an important general cellular function. Early studies on vaults focused on the structural features and cellular distribution of the particle and will only be summarized briefly here. In this article, we discuss the molecular characterization of vault components and describe genetic studies carried out in Dictyostelium. The recent finding that the major vault protein is elevated in non-P-glycoprotein multidrug resistant cancer cells has direct implications concerning the function of the vault particle and indicates a potential role for vaults in resistance of tumour cells to anticancer drugs.