Properties of a GTP sensitive microdomain in rough microsomes.

Stripped rough microsomes (SRM) fuse when incubated with physiological concentrations of GTP and MgCl2. In order to examine further to what extent such fusions are associated with other membrane functions of rough endoplasmic reticulum, we have evaluated the role of cytosolically exposed peptide constituents of SRM in fusion, and the possible relationship of GTP/MgCl2-induced fusion in protein transport across endoplasmic reticulum (ER) membranes, and in ER-Golgi interactions. Controlled proteolytic digestion of SRM led to the loss of fusion capability at 15 micrograms/ml trypsin--a concentration which maintained the latency of intraluminal mannose-6-phosphatase. Hence, a cytosolically exposed protein(s) regulated fusion. Based on ribonuclease-induced ribosome capping experiments, it was further concluded that the cytosolic oriented protein(s) was sequestered beneath the ribosome. As co-translational cell free translocation of placental lactogen across SRM was similar in control membranes compared to those rendered incapable of fusing, it was concluded that the fusion phenomenon may not be related to translocation. Under conditions promoting homologous fusion of SRM or Golgi membranes, mixtures of the two membranes showed no heterologous membrane fusion as assessed morphologically or by the transport of newly synthesized membrane glycoprotein. These experiments attest to the specificity of cytosolically exposed protein(s) in regulating nucleotide/divalent cation-induced membrane fusion.

Tunicamycin sensitivity of prolactin, insulin and epidermal growth factor receptors in rat liver plasmalemma.

We have used the glycosylation inhibitor tunicamycin to assess the stability of the receptors for prolactin, insulin and epidermal growth factor (EGF) in rat liver cell membrane. Direct binding studies on liver plasmalemma fractions which were isolated from tunicamycin-treated rats revealed a rapid loss of prolactin receptors (t1/2 approximately 35 min) with a more prolonged half-life for insulin (10 h) and EGF receptors (8 h). The rates of receptor loss were similar to the respective half-lives of the receptors as documented by others using cultured cells. The respective ligands for each receptor were lost more rapidly from liver, i.e. prolactin, t1/2 approximately 10 min, insulin, t1/2 approximately 5 min and EGF, t1/2 approximately 17 min. Previous studies have shown ligand loss in vivo to be receptor mediated. Thus, receptors and their ligands do not turn over synchronously in vivo. These studies also point to a major role for N-linked oligosaccharide side chains in the functional insertion of prolactin, insulin and EGF receptors into the hepatocyte cell surface in vivo.

Towards a European consensus on conducting and reporting health economic evaluations--a report from the ISPOR Inaugural European Conference.

This report is a summary of key issues in consensus development regarding the conduct and reporting of health economic research in the European context, presented and discussed at the ISPOR Inaugural European Conference in Cologne, Germany, December 1998. Recommendations of the Harmonization by Consensus of the Methodology for Economic Evaluation of Health Care Technologies in the European Union (HARMET) project were presented, as well as two instruments under development: software for Reporting Economic Evaluation Results (REER) and software for collecting and managing cost data called the Health Cost Database Software (HCDS). Working independently, but interrelated with the objectives of the HARMET initiative, preliminary results from the ongoing European Network on Methodology and Application of Economic Evaluation Techniques (EUROMET) project were presented. Each presentation was followed by an expert discussion panel with audience participation. Issues raised included the development of standards and related topics such as usefulness to European decision-makers, and education and training in health economics in Europe.

Organelle-specific phosphorylation. Identification of unique membrane phosphoproteins of the endoplasmic reticulum and endosomal apparatus.

Highly purified endoplasmic reticulum fractions from rat liver and dog pancreas harbor membrane-associated kinases that phosphorylate integral membrane proteins of 90, 56, 35, and 15 kDa with [gamma-32P]GTP and of 90, 56, and 35 kDa with [gamma-32P]ATP. Of these, only the 35-kDa phosphoprotein was N-glycosylated. Screening of Golgi fractions, endosomes, plasma membranes, lysosomes, and mitochondria revealed phosphoproteins unique to each organelle. In particular, endosomes were found to harbor a 48-kDa extrinsic membrane protein and two or more integral membrane phosphoproteins of 30-35 kDa. None of these were N-glycosylated as judged by their insensitivity to digestion by N-glycosidase F and a lack of binding to concanavalin A or wheat germ agglutinin. Since the 30-35 kDa membrane phosphoproteins present in Golgi-free endosomal fractions were not detected in endosome-free, highly purified Golgi fractions and were found exclusively in horseradish peroxidase-containing endosomes as determined by the diaminobenzidine shift protocol, then these membrane phosphoproteins are unique to endosomes. Since membrane phosphoproteins unique to the endoplasmic reticulum have been shown to have important functional significance in calcium binding and as a membrane chaperone(s) (Wada, I., Rindress, D., Cameron, P.H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D.Y., and Bergeron, J.J.M. (1991) J. Biol. Chem. 266, 19599-19610; Ahluwalia, N., Bergeron, J.J.M., Wada, I., Degen, E., and Williams, D.B. (1992) J. Biol. Chem. 267, 10914-10918), then the unique endosomal phosphoproteins may serve equally important functions in addition to serving as novel markers for the organelle.

SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane.

GTP phosphorylation of rough microsomes in vitro is limited to four integral membrane proteins. Two of these, a phosphoprotein (pp90) and a phosphoglycoprotein (pgp35) were purified as a complex with two nonphosphorylated membrane glycoproteins, gp25H and gp25L. The authenticity of this complex was confirmed using two different purification procedures and by coimmunoprecipitation. By immunofluorescence a reticulated cytoplasmic network was revealed for the proteins which was similar to that for Louvard et al. (Louvard, D., Reggio, H., and Warren, G. (1982) J. Cell Biol. 92, 92-107) marker antisera which also recognized purified pp90 on immunoblots. Amino acid sequencing of peptides derived from pgp35 identified this protein as SSR alpha, an endoplasmic reticulum constituent as identified by cross-linking of translocating nascent chains (Görlich, D, Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., and Rapoport, T. A. (1990) J. Cell Biol. 111, 2283-2294). The sequence of gp25H was determined from cDNA clones and was identical with SSR beta identified by Görlich et al. (1990) as being tightly bound to SSR alpha. Sequencing of gp25L revealed no similarity of the deduced sequence with other proteins. However, pp90 revealed a high degree of sequence identity with the Ca(2+)-binding protein, calreticulin. 45Ca2+ overlay studies indicated that pp90 bound Ca2+ and the name calnexin is proposed. Surprisingly, pgp25 (SSR alpha) also bound Ca2+ although gp25H (SSR beta) and gp25L did not. Triton X-114 partitioning of the integral membrane proteins of rough microsomes suggested that pgp35 (SSR alpha) and calnexin were major Ca(2+)-binding proteins of the endoplasmic reticulum membrane. We propose that the function of the complex is to regulate Ca(2+)-dependent retention mechanisms for luminal proteins of the endoplasmic reticulum.