The goals are to define the pathways leading to hepatic secretion and intracellular trafficking of proteins and to begin to understand the organization and expression of genes specifying serum proteins. This proposal describes plans for the production, isolation, and characterization of mutants of rat hepatoma cells that exhibit temperature dependent secretion (Sec-ts) of albumin (RSA) and Alpha-1-antitrypsin (A1AT) and another class of mutants that secrete different sets of serum proteins. To isolate Sec-ts mutants, mutagenized rat hepatoma cells that grow at 33.5 degrees but not at 39 degrees will be selected and enriched for secretory mutants. Colonies of these cells will be grown at 33.5 degrees, shifted to 39 degrees and then screened by a filter immunoassay coupled with an in situ hybridization technique to identify clones that do not secrete RSA and/or A1AT at 39 degrees but do contain mRNAs specifying these proteins. Clones of this type will be further examined to identify those that synthesize but fail to secrete either or both of these proteins at the non-permissive temperature. These mutants will be characterized by genetic, immunological, and biochemical techniques to establish the number of genes specifying components of the secretory pathway and to identify individual components of the intracellular trafficking and secretory pathways. Monoclonal antibodies that recognize intracellular membranous components will be developed and used in in vivo and in vitro studies to aid in defining these pathways. To study regulation of expression of genes specifying serum proteins mutants of rat hepatoma cells that secrete some but not all of the serum proteins will be identified by the filter immunoassay and analyzed to estabish whether or not sets of these genes may be coordinately regulated. For example, clones that secrete transferrin but not albumin, and visa versa, will be isolated and the serum proteins secreted from these cells identified by crossed-immunoelectrophoresis and two dimensional electrophoresis. The objective is to identify sets of serum proteins whose secretion is consistently linked to the secretion of albumin or transferrin. Screening for mutants will be expanded to identify cells that secrete one or the other of different combinations of serum proteins such as RSA and A1AT, A1AT and transferrin, Alpha2-macroglobulin and A1AT, etc. These studies will show which of the serum proteins are secreted as distinct sets and thus indicate which genes may be coordinately regulated. Successful completion of the experiments described in this proposal will lead to a better understanding of the mechanisms involved in secretion and intracellular trafficking of proteins in liver and will provide an insight into the organization and means of control of genes specifying serum proteins. In addition, certain of the Sec-ts mutants may prove to be valuable models for studying the molecular basis for the human disease A1At deficiency.
| Henry 2nd, K W; Brown, J L; McCracken, A A (1990) Selective enrichment for temperature-sensitive secretion mutants of mammalian cells using plant lectin, concanavalin A. Somat Cell Mol Genet 16:297-304 |
