Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

Immunofluorescence studies indicated that for mutants 42-61, 43-61, and 47-6 l, VP7 staining co-localized with that of elements of the Golgi apparatus in COS 7 cells, indicating that these VP7 mutants were probably transported out of the ER. Therefore, the incubation media from all the transfected cell cultures described above were examined for secreted VP7 products. Three of the deletion mutants, 42-61, 43-6 l, and 47-61, affecting the distal region of the second hydrophobic domain produced VP7 molecules which were secreted (Fig. 7) , in marked contrast to the behavior of the wild-type VP7 (pJC9), and the other mutants (51-61 in particular) (Fig. 7) . The latter differed by only four amino acids from the secreted 2206 THE JOURNAL OF CELL BIOLOGY • VOLUME 101, 1985 mutant 47-61 (Fig. 2 ). In addition, the secreted VP7 products were endo-H resistant (Fig. 7, + lanes) , consistent with their passage through the Golgi apparatus and their modification to the complex type of carbohydrate. Densitometry of respective bands of original autoradiographs used for Figs. 6 and 7, which were equivalently exposed, showed that similar amounts of secreted and intracellular VP7 were present. These results pertain to cells labeled for 4 h. However, similar results were obtained in a 2.5-h labeling period (data not shown), indicating that secretion is an efficient process. The size of the VP7 which contained complex oligosaccharides (VP7 c) was slightly larger than that of VP7 with the high-mannose form of glycosylation (VP7) (Fig. 8, lanes 5 and 7) as expected from the known higher molecular weight of complex N-linked oligosaccharides. Mutant 42-61 also secretes, based on its size, a nonglycosylated version of its mutant VP7 protein (Fig. 7) . This was confirmed by another experiment where cultures transfected with mutants 42-6 l, 43-6 l, or 47-61 were subsequently treated with tunicamycin ( Fig. 8 , mutant 43-6 l only shown). The product generated in the presence of tunicamycin (Fig. 8 , + lanes) (VP7 u) is identical in size to the naturally occurring unglycosylated product of mutant 42-6 l (Fig. 7) . Some material the size of VP7 remained after tunicamycin treatment (Fig. 8, lane 8) probably because of limiting amounts of the drug, since in other experiments tunicamycin resulted in complete conversion to VP7 u. In control cells, those not treated with tunicamycin (Fig. 8, lane 5) , the endo-H-resistant product was again secreted into the medium. Similar results were obtained with mutants 42-61 and 47-61 (data not shown). The amount of protein secreted in tunicamycin-treated cells (Fig. 8, lane 6) was lower than that of the control, possibly because the drug interferes with secretion. No such bands were identified when the vector (pJC 119), without VP7 gene insert, was used for transfection (Fig. 8, lanes 1-4) . The presence of prominent nonspecific background bands (Fig. 8, lanes 1, 2, 4, and 6-8) , which are different for the intracellular (intra) and secreted (sec) lanes, does not alter this conclusion. These results confirm that the FIGURE 7 Endo-H sensitivity of immunoprecipitated products from the media of cells transfected with wild-type and mutated VP7 genes. Cells were labeled for 4 h with L-[3SS]methionine as described, the medium from each culture subjected to immunoprecipitation (-lanes), and half of the samples digested with endo-H (+ lanes). Total SA11-infected MA104 cell lysate before (-) and after (+) Endo-H displays marker glycosylat