PMC

The helix α9 ERp29 mutants do not significantly affect Tg secretion. Mock-transfected FRTL-5 cells or cells overexpressing WT, L240K, I246K, or L247K ERp29 were labeled metabolically. Secreted Tg was immunoprecipitated from the culture media, and the immune complexes were analyzed by SDS-PAGE followed by autoradiography to detect Tg and by immunoblotting with an ERp29 antibody to detect ERp29 (data not shown). The amount of secreted Tg was quantified and normalized to that of ERp29 expression. Data are means ± standard errors.

The helix α9 ERp29 mutants bind less efficiently than WT ERp29 to PyV. PyV was incubated with extracts from cells expressing WT, L240A, I246A, or L247A ERp29. The cross-linking reagent DSP was added to the reaction mixtures. After the reaction was quenched, the WT and mutant ERp29 proteins were immunoprecipitated with an ERp29 antibody or a control Myc antibody, and the immune complexes were analyzed by reducing SDS-PAGE followed by immunoblotting with an antibody against ERp29 or against VP1 to detect PyV. The asterisk (*) represents an unidentified protein that is precipitated by the Myc antibody. IP, immunoprecipitation.

The helix α9 ERp29 mutants are not globally misfolded. (A) The helix α9 ERp29 alanine mutants migrate similarly to WT ERp29 by size exclusion chromatography. Extracts from cells overexpressing WT, L240A, I246A, or L247A ERp29 were individually subjected to size exclusion column chromatography. The column was washed isotonically with a physiological buffer containing no sucrose. Fractions were continuously collected from the column, and the even-numbered fractions were analyzed by reducing SDS-PAGE followed by immunoblotting with an antibody against ERp29. The column was calibrated using a gel filtration standard. (B) The helix α9 ERp29 alanine mutants dimerize efficiently. Extracts from NIH 3T3 cells overexpressing WT, L240A, I246A, or L247A ERp29 were incubated with various concentrations of the cross-linking reagent DSP. The cross-linking reaction was quenched, and the samples were analyzed by nonreducing SDS-PAGE followed by immunoblotting with an antibody against ERp29. (C) The helix α9 ERp29 alanine mutants have protease sensitivity similar to that of WT ERp29. Extracts from cells overexpressing WT, L240A, I246A, or L247A ERp29 were incubated with various concentrations of trypsin. The digestion reactions were quenched, and the samples were analyzed by reducing SDS-PAGE followed by immunoblotting with an antibody against ERp29.

Effects of the helix α9 ERp29 mutants on PyV unfolding and infection. (A) I246K and L247K ERp29 do not unfold VP1. PyV was preincubated with extracts from mock-transfected NIH 3T3 cells or from cells overexpressing WT, L244K, I246K, or L247K ERp29. Trypsin was then added to the reaction mixtures. The samples were analyzed by reducing SDS-PAGE and immunoblotting with antibodies against VP1 (top panel) or ERp29 (bottom panel). (B) I246K and L247K ERp29 stimulate PyV infection inefficiently. Mock-transfected NIH 3T3 cells and cells overexpressing WT, I246K, or L247K ERp29 were challenged with PyV (100 PFU/cell). Large-T-antigen expression was analyzed by standard fluorescence microscopy. (C) Alanine mutations at positions 240, 246, and 247 in helix α9 abrogate ERp29's unfolding activity. The experiment was the same as for panel A, except that extracts from cells overexpressing L240A, I246A, and L247A ERp29 were used. (D) Helix α9 alanine mutants of ERp29 do not stimulate PyV infection. The experiment was the same as for panel B, except that cells overexpressing L240A, I246A, and L247A ERp29 were used. Data are means ± standard deviations.

The L240K ERp29 mutant does not stimulate PyV unfolding and infection efficiently. (A) DTT and EGTA destabilize PyV. PyV was incubated with DTT and EGTA where indicated, followed by the addition of trypsin. The samples were subjected to reducing SDS-PAGE followed by immunoblotting with an antibody against VP1. (B) L240K ERp29 cannot unfold VP1. PyV was preincubated with extracts from mock-transfected NIH 3T3 cells or from cells overexpressing WT, L240K, K226E, L227S, E220Q, or R223A ERp29. Trypsin was then added to the reaction mixtures. The samples were analyzed by reducing SDS-PAGE and immunoblotting with antibodies against VP1 (top panel) or ERp29 (bottom panel). The asterisk (*) represents a partial degradation product of VP1a. (C) L240K ERp29 stimulates PyV infection inefficiently. Mock-transfected NIH 3T3 cells and cells overexpressing WT or L240K ERp29 were challenged with PyV (100 PFU/cell). Large-T-antigen expression was analyzed by standard fluorescence microscopy. (D) The experiment was the same as for panel C, except that cells were challenged with 200 PFU/cell. Data are means ± standard deviations.