Abstract. Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of •6.2 which we have designated as nupl80. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGASepharose demonstrated that nupl80 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nupl80 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nupl80 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nupl80 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.

Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for an in organello site-directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial signal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety. While this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the endogenous mitochondrial signal peptide processing machinery as well as the cleavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptide-DNA-conjugates were treated with purified mitochondrial intermediate peptidase. When the leader peptide is directly linked to the DNA moiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid residues of the mature part of OTC are appended at the carboxy-terminal end of the signal peptide. Our results show that artificial peptide-DNA-conjugates are recognized by the mitochondrial proteolytic machinery, and therefore an interference of the peptide with the DNA function can be excluded.

Background information. During early phases of Xenopus oogenesis, 5S rRNA and tRNAs are stored in the cytoplasm of young oocytes in the form of a common RNA-protein complex termed the 42S particle. These storage particles comprise two kinds of proteins with different RNA binding specificities. The tRNA-binding protein 42Sp50 belongs to the EF1A (eukaryotic translation elongation factor 1A) family of translation elongation factors, while 42Sp43 is a diverged form of the transcription factor TFIIIA (transcription factor IIIA) and binds 5S rRNA. Little is known about the mode of protein-protein interactions that stabilize the 42S particle.Results. We have determined the intracellular localization of the protein components of the 42S particle by expressing fluorescent protein-tagged fusions in transparent previtellogenic oocytes. 42Sp50 and its isoforms (EF1A-S and EF1A-O) were excluded from the nuclei and distributed uniformly throughout the cytoplasm with no enrichment in the Balbiani bodies, as described earlier by immunocytochemistry. In contrast, 42Sp43 accumulated in the amplified nucleoli. However, when both proteins were simultaneously expressed, 42Sp43 was no longer present in the nucleoli but was retained, together with 42Sp50, in the cytoplasm, the most likely site of 42S particle assembly. In contrast, the somatic-type EF1A isoforms were unable to redirect 42Sp43 from the nucleolar to the cytoplasmic compartment. We also tested for in vivo interactions using transiently transfected mammalian cells (COS-7 cell line). In this heterologous cell system 42Sp43 remained bound to the nucleoli but, on co-expression, induced the redistribution of 42Sp50 from the cytoplasm to the nucleoli.Conclusions. The microscopic approach described allows visualization of protein-protein interactions involved in the assembly of 42S storage particles. In particular, the transfection assay using COS-7 cells provides a rapid screening test that should facilitate identification of critical residues and structural determinants that enable the proteins of the 42S storage particle to interact with each other and to establish distinct higher-order RNP (ribonucleoprotein) complexes.

No abstract

No abstract

No abstract

No abstract