Highlights d Human cells release Argonaute 1-4 and major vault protein independently of exosomes d Annexin A1 is a specific marker of microvesicles shed from the plasma membrane d Small extracellular vesicles do not contain DNA d Active secretion of cytosolic DNA occurs through an amphisome-dependent mechanism

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

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.

Abstract. Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 x 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: M, 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for >70% of the total Coomassie Brilliant Blue-staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing ~,,140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains ,,,,9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (M, 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32p-labeled RNA of identical size from metabolically-labeled rat hepatoma ceils. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown.IBONUCLEOPROTEIN structures have recently been demonstrated to be of greater diversity and significance than previously realized. Although the importance of the ribosome in cellular metabolism is well established, the functional roles of the small nuclear ribonucleoprotein particles are only currently being explored (2, 12; for review see references 6 and 9). Recently it has also been demonstrated that the targeting of nascent secretory polypeptides to the endoplasmic reticulum is mediated by an llS structure termed signal recognition particle (29) which contains a 7S RNA essential for its function (30). Another small (10S) cytoplasmic ribonucleoprotein particle is apparently involved in the regulation of protein synthesis (25). Each of these small ribonucleoprotein particles contains multiple polypeptides and a single species of RNA.We describe the purification of a novel ribonucleoprotein particle from rat liver microsomal extracts. This structure is unusual in its size, molecular composition, and morphology. It is considerably larger than the ribosome and is therefore the largest ribonucleoprotein body reported to date. Its protei...