Intracellular localization of the HCS2 gene products in identified snail neurons in vivo and in vitro.

1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca(2+) concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy. 2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system. 3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons. 4. The immunogold particles were practically absent in the bodies of cultured neurons. 5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.

Compartmentalization of certain components of the protein synthesis apparatus in mammalian cells.

A comparative study on the localization of free cytosolic tryptophanyl-tRNA synthetase (TrpRS) and several components of the multi-aminoacyl-tRNA synthetase (ARS) complex (glutamyl-prolyl-tRNA synthetase (GluProRS), arginyl-tRNA synthetase (ArgRS)), and two non-synthetase polypeptides p38 and p43 has been carried out on ultrathin sections of cultured rabbit kidney cells by the immunogold technique using monoclonal antibodies raised against appropriate polypeptides. It has been shown that GluProRS, ArgRS and p38 polypeptide are distributed in the cells similarly to TrpRS and are located mainly in the vicinity of ribosomes. A smaller but significant portion of these proteins has been observed in the nuclei in the diffuse chromatin regions and in the vicinity of interchromatin granules. On the contrary, the main part of p43 protein was found in the cell nuclei; this indicates that this protein may exist in the cell separately from the cytoplasmic multi-ARS complex. Our results argue in favor of compartmentalization of both free ARS (such as TrpRS) and the multi-ARS complex in the vicinity of ribosomes. At the same time, the detection of some ARS in the diffuse chromatin regions in the nucleus implies that these enzymes may exhibit some non-canonical functions in addition to their role in protein synthesis.

Immunoelectron microscopic location of tryptophanyl-tRNA synthetase in mammalian, prokaryotic and archaebacterial cells.

Monoclonal antibody Am1 against conservative epitope of tryptophanyl-tRNA synthetase (WRS) was labeled with colloidal gold particles and used to localize the enzyme on ultrathin sections of eubacteria (Escherichia coli), archaebacteria (Methanococcus halophilus), rat pancreas tissue and rat fibroblasts (cell line RAT1). In all cell types immunoelectron microscopy revealed predominant cytoplasmic location of gold particles, as this could be expected from known biochemical data. In particular, in mammalian cells intensive labeling was observed in cytoplasmic regions rich in polysomes and free ribosomes. At the same time, the label was virtually absent in cytoplasmic regions where microfilament bundles were present. Significant concentrations of gold particles were found in mitochondria and nuclei. In the latter case, gold particles were located over diffuse chromatin regions and were virtually absent over compact chromatin. The density of diffuse chromatin in labeling may amount to about 50% of that found in the cytoplasm. Distribution of labeled antibodies over E. coli cells looks rather similar to that found for M. halophilus: gold particles are preferably concentrated over the cytoplasm and "boundary zone", i.e., a 30 nm wide cytoplasmic zone adjacent to the nucleoid border, while the label over nucleoid is virtually absent. Two main conclusions are drawn: (i) although in the animal cell homogenates WRS is recovered mainly as a soluble cytosolic enzyme, in intact cells it is associated with defined cellular organelles and compartments; this may be an evolutionarily acquired feature probably typical for multicellular organisms; (ii) the considerable density of labeling in diffuse (not compact) chromatin regions may be indicative of WRS involvement in the active chromatin functions (transcription, processing, transfer of gene products, etc.).