TABLE 2.
Oligomerization and trypsin sensitivity of G protein mutantsa
| Protein | Trimer stability at pH 5.6b | Trypsin resistance atc:
|
||
|---|---|---|---|---|
| pH 7.4 | pH 6.5 | pH 5.6 | ||
| WT | + | − | ++ | +++ |
| W1A | + | − | ++ | +++ |
| W2A | + | − | ++ | +++ |
| WW-AA | + | − | ++ | +++ |
| DAA | + | − | ++ | +++ |
| G(AXB) | + | − | ++ | +++ |
| GSrev11 | + | − | ++ | +++ |
| GSrev11-AA | + | − | ++ | +++ |
| GΔ9 | + | − | + | +++ |
| GΔ13 | + | − | ++ | +++ |
| ΔF440-N449 | + | − | ++ | +++ |
| G10DAF | + | − | + | +++ |
| GΔ9-10DAF | + | − | ++ | +++ |
| G(+9)gBG | ± | − | − | ++ |
Confluent monolayers of BHK-21 cells were infected with either WT or mutant viruses at a multiplicity of 10. At 6 h postinfection, the cells were radioactively labeled and chased as described in Materials and Methods.
For the trimer assays, the cells were lysed in 2× MNT buffered to pH 5.6. Lysates were then centrifuged through a 5 to 20% sucrose density gradient buffered to the same pH. Fractions were collected from the bottom, and G proteins were immunoprecipitated with a polyclonal anti-VSV antiserum.
For the trypsin sensitivity assays, cells were lysed in 1× MNT buffered to the indicated pH. Lysates were clarified by centrifugation to remove cell debris and nuclei. The supernatants were then treated with or without TPCK-trypsin, and the G proteins were immunoprecipitated with a polyclonal anti-VSV antibody. Immunoprecipitated proteins were resolved by SDS-PAGE, visualized by fluorography, and quantified by scanning densitometry. +++, 95 to 100% resistant; ++, 50 to 95% resistant; +, 10 to 50% resistant; −, <10% resistant.