Gap junctions are complexes between adjacent cells that are responsible for direct intercellular communication via passive diffusion of low-molecular-weight hydrophilic molecules. The proteins present in gap junctions are termed connexins, of which at least 16 distinct gene products are known to occur in vertebrate species. 1,2 Connexin proteins differ notably from other membrane proteins in lacking glycosylation and being localized primarily at appositional regions between coupled cells. While the pathways of biogenesis, trafficking, assembly, and turnover have not yet been completely elucidated, key features appear to be more similar than not to ''typical'' plasma membrane proteins. [3][4][5][6][7][8][9][10][11] In previous studies, biochemical analysis of secretory or trafficking mutants 12-14 has elucidated many features of these pathways. This approach for analysis of gap junction biology has not been pursued in vertebrate cell lines harboring similar mutations. Characterization of connexin trafficking in the membrane protein trafficking mutant, Trf1, was undertaken as a first step in combining biochemical and mutational approaches to analysis of connexin trafficking and assembly.The Trf1 cell line was isolated as a result of a dual selection protocol. 15 Endocytic activity of 2 different carbohydratespecific receptors of the human hepatoma cell line HuH-7 were simultaneously selected against using gelonin, an inhibitor of protein synthesis, conjugated to galactose-and mannoseterminating glycoproteins. Because there was no significant reciprocal inhibition between the mannose-and galactoseterminating glycoproteins and their respective gelonin conjugates, the mutation harbored by Trf1 cells appeared to affect a common step in their endocytic pathway. This pleiotropic phenotype was extended to other cell-surface membrane proteins, including the transferrin receptor and the phenylalanine transporter.The Trf1 mutation provided the first genetic evidence for the existence of different subpopulations (States 1 and 2) of the asialoglycoprotein receptor (ASGPR) as described by Weigel and Oka. 16 Originally based on the biphasic kinetics of ligand-receptor complex dissociation, numerous characteristics have since been described that differentiate the 2 ASGPR subpopulations and the proposed endocytic pathways that they mediate. 17 While no biochemical basis for this receptor dichotomy has been established, only State 2 receptors were shown to be susceptible to modulation by a wide variety of cell perturbants and altered metabolic states of the liver. 17 The proposed existence of 2 subpopulations of receptors is not limited to ASGPR and has been suggested for a wide variety of cell-surface receptors on different cell types. The low-density lipoprotein and mannose-6-P receptors on fibroblasts, the mannose receptor and ␣ 2 macroglobulin receptor on alveolar macrophages, and the transferrin receptor on K562 and HuH-7 cells all respond to cell perturbants by a partial reduction in their surface expression, suggesting Abb...
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