Yutaka Tashiro

Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry

The isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes were characterized by lectin cytochemistry using concanavalin A (ConA), Ricinus communis agglutinin 120 (RCA-120), and wheat germ agglutinin (WGA). We found that RCA-120, ConA, and WGA bind to these membranes. The distribution of the lectins on the isolation membranes was heterogeneous, mainly found on the rims, which we referred to as the peripheral dilated portion. When the rims fused and thus formed autophagosomes the apparent sites of fusion were strongly labeled by the lectins. After autophagosomes were transformed to autolysosomes by fusion with lysosomes, the limiting membranes became more densely and homogeneously labeled with the lectins. We previously reported that cytochrome P-450 does not exist on the limiting membranes of the autophagosomes. Taken together, these results suggest that the isolation membranes may originate not from endoplasmic reticulum membranes but from some post-Golgi membranes that contain complex type N and/or O-linked oligosaccharide chains.

Heavy chain binding protein (BiP/GRP78) and endoplasmin are exported from the endoplasmic reticulum in rat exocrine pancreatic cells, similar to protein disulfide-isomerase

Previously we found that in rat exocrine pancreatic cells, protein disulfide-isomerase (PDI), one of the major resident proteins in the lumen of the endoplasmic reticulum (ER) of many cells, is localized not only in the ER but also in the Golgi apparatus, secretory granules, plasma membranes, and even in the glandular lumens, despite possessing the ER retention signal KDEL (Lys-Asp-Glu-Leu) at the carboxyl terminus. In this report, we examined whether other ER luminal proteins bearing the KDEL signal at their C-termini, such as BiP/GRP78 and endoplasmin/GRP94 are also exported from the ER. We prepared two kinds of affinity-purified polyclonal antibodies; one against a synthetic peptide with 12 amino acids which is identical to the carboxyl terminus of BiP and another against purified endoplasmin. Immunoblot analysis using these two antibodies showed that BiP and endoplasmin exist in both the plasma membrane and the microsomal fractions, similar to the intracellular distribution of PDI in rat exocrine pancreas. The ratios of the amount of the three proteins in the two fractions, however, were variable, suggesting that the KDEL-bearing proteins such as PDI, BiP, and endoplasmin are exported from the ER with different efficiencies. Postembedding protein A-immunogold electron microscopy revealed that endoplasmin was exported from the ER and secreted to the extracellular space. The secretion of PDI in rat pancreatic lobules was inhibited by Brefeldin A (BFA) and by guanidino acid esters (FOY-305), which are known to be the inhibitors of the intracellular transport. Taken together with the previous immunogold electron microscopic analyses by Akagi et al. (1988), it is strongly suggested that in rat exocrine pancreatic cells PDI and the other KDEL-bearing proteins found in the extracellular space were not artificially released by cell damage during incubation but were secreted via the normal secretory pathway.

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissners membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.

Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

Association of N‐ethylmaleimide‐sensitive factor with synaptic vesicles

N‐Ethylmaleimide‐sensitive factor (NSF) mediates docking and/or fusion of transport vesicles in the multi‐pathways of vesicular transport. NSF was highly expressed in brain and adrenal gland. Immunostaining of cerebellum with an anti‐NSF monoclonal antibody showed that NSF is predominantly localized in the molecular layers and the glomeruli of the granule cell layers. This distribution coincided well with that of synaptophysin, a marker protein of synaptic vesicles. Purification and immunoprecipitation revealed that NSF is associated with brain synaptic vesicles. The present results suggest that NSF is associated with synaptic vesicles without Ca2+ influx.

Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells.

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.

Distribution of protein disulfide isomerase in rat hepatocytes.

We investigated quantitatively the distribution of protein disulfide isomerase (PDI) in rat hepatocytes by immunocytochemistry using a post-embedding protein A-gold technique. In hepatocytes, gold particles were mainly localized in the intracisternal space of the rough and smooth endoplasmic reticulum (ER) and nuclear envelopes. Autolysosomes engulfing ER were occasionally densely labeled, especially in rat hepatocytes previously treated with leupeptin in vivo, suggesting that the autophagosome-autolysosome system may be an important route for degradation of PDI. A few gold particles were also found on the plasma membranes. Localization of gold particles on the other subcellular organelles, such as Golgi apparatus, peroxisomes, and nuclear matrix, was sparse and at the control level. The predominant localization of PDI on the intracisternal surface of the ER and nuclear envelope supports a potential role of PDI in the formation of disulfide bonds of nascent polypeptides, thus accelerating formation of the higher-order structure of secretory and membrane proteins and rendering the translocation process irreversible.

Sedimentation studies on the interaction of ribosomal subunits from liver

Abstract 1. The 50-S and 60-S components which appear in the intermediate stage of dissociation of hepatic ribosomes were isolated by sucrose density-gradient centrifugation. Sedimentation analyses have shown that these two components are each composed of large and small subunits. 2. The release of the small subunit from the isolated 50-S and 60-S components was found to be exclusively dependent on Mg2+ concentration. 3. Stable small and large subunits were isolated in the presence of 1 mM Mg2+ and 0.1 mM Mg2+, respectively. Association of the isolated small subunit as well as of the isolated large subunit was observed in the presence of more than 2 mM Mg2+. It has not been possible to show any preferential association between the small subunit particles as compared with the large subunit particles, nor has any selective interaction between the small and the large subunit particles been demonstrated.

Correlation between phospholipase A2 activity and intra-Golgi protein transport reconstituted in a cell-free system

A wide variety of phospholipase A2 inhibitors blocks intra‐Golgi protein transport reconstituted in a cell‐free system. Phospholipase A2 activity detectable under the protein transport assay conditions is actually inhibited by the inhibitors. There is a good correlation between the inhibition of protein transport and that of phospholipase A2 activity. Prolactin secretion from GH3 cells is also blocked by a membrane‐permeable phospholipase A2, inhibitor, suggesting the physiological relevance to inhibition of protein transport in vitro by phospholipase A2 inhibitors.

Sedimentation analyses of native silk fibroin in urea and guanidine·HCl

Abstract 1. 1.|Sedimentation analyses of native silk fibroin were carried out in 0–6 M urea and guanidine·HCl. It is suggested that the native 10-S fibroin molecules are not dissociated into subunits in these denaturing agents. 2. 2.|Addition of dithiothreitol to the fibroin solution resulted in release of minor components and appearance of a major homogeneous component (6.8-S component). The decrease in the s value from 10 to 6.8 S depends markedly on the concentration of urea or guanidine·HCl. In urea solution of more than 3.0 M or in guanidine · HCl solution of more than 1.0 M, hardly any decrease in the sedimentation coefficient of the major homogeneous component was observed. From the s value in 6.0 M guanidine·HCl, molecular weight of the major homogeneous component was estimated to be about 2.5·105. 3. 3.|Molecular weight of main or 10-S component of the native silk fibroin, 6.8-S component of reduced fibroin and major component of reduced and carboxymethylated fibroin determined in 6.0 M guanidine·HCl by a sedimentation equilibrium method was 3.7·105 ± 0.2·105, 3.0·105 ± 0.2·105 and 3.1·105, respectively. 4. 4.|From these results it is suggested that the native fibroin molecules are composed of minor heterogeneous low molecular weight components and major homogeneous component probably connected together by disulphide bonds and that the latter is composed of either a single polypeptide chain or several polypeptide chains bound by covalent bonds other than disulphide bonds.