All domains of Cry1A toxins insert into insect brush border membranes

Abstract

A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only alpha-helix 5 of domain I along with adjacent helices alpha-4 or alpha-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of alpha-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.

FIGURE 1.

A, Western blot analysis of purified Cry1A toxin used for proteinase K protection assays. Lane 1, Cry1Aa; lane 2, Cry1Ab; lane 3, Cry1AbL40C; lane 4, Cry1AaD62C; lane 5, Cry1AbV171C; lane 6, Cry1AbS191C; lane 7, Cry1AbL199C; lane 8, Cry1AbL215C. B, Western blot analysis of purified Cry1A toxin used for proteinase K protection assays. Lane 1, Cry1AbS279C; lane 2, Cry1AbS324C; lane 3, Cry1AbS364C; lane 4, Cry1AbF371W; lane 5, Cry1AbF371C; lane 6, Cry1AaE460C; lane 7, Cry1AaK489C; lane 8, Cry1AaI526C. C, purified domain I mutant proteins run on 8% SDS-polyacrylamide gel. Lane 1, protein standard; lane 2, 1Ab L40C; lane 3, 1Aa D62C; lane 4, 1Ab V171C; lane 5, 1AbS191C; lane 6, 1Ab L199C; lane 7, 1Ab L215C. D, purified domain II and domain III mutants run on 8% SDS-polyacrylamide gels. Lanes 1 and 9, protein standards; lane 2, 1Ab S279C; lane 3, 1Ab S324C; lane 4, 1Ab S364C; lane 5, 1Ab F371C; lane 6, 1AaE460C; lane 7, 1Aa K489C; lane 8, 1Aa I526C.

FIGURE 2.

A, Western blot analysis of 8% SDS-PAGE run of proteinase K protection assay of Cry1A mutants bound to BBMV. The mutants used are as follows: lane 1, Cry1Ab; lane 2, 1AbL40C; lane 3, 1AaD62C; lane 4, 1AbV171C; lane 5, 1AbS191C; lane 6, 1AbL199C; lane 7, 1AbL215C. B, Western blot analysis of 8% SDS-PAGE run of proteinase K protection assay of Cry1A mutants bound to BBMV. The mutants used are as follows: lane 1, Cry1Aa; lane 2, 1AbS279C; lane 3, 1AbS324C; lane 4, 1AbS364C; lane 5, 1AbF371W; lane 6, 1AbF371C;lane 7, 1AaE460C; lane 8, 1AaK489C; lane 9, 1AaI526C.

FIGURE 3.

Percentage of quenching of fluorescence of 1,5-IAEDANS-tagged cysteine mutants calculated as (IIaq – ISUV)/Iaq, where Iaq is the quantum yield of fluorescence of the labeled mutants in aqueous buffer, and ISUV is the quantum yield of fluorescence of the labeled mutants in SUV.

FIGURE 4.

Blue shift in the maximal emission wavelength of each acrylodan-labeled mutant. The x axis indicates the name of each labeled mutant studied, and the y axis shows the maximal emission wavelength. Maximal emission wavelength of each mutant in aqueous carbonate buffer is indicated by •, in BBMV without any protease treatment is indicated by ○, and in BBMV after proteinase K treatment is indicated by ▾.

FIGURE 5.

Steady state fluorescence measurement for the following acrylodan-labeled mutants. A, D62C (domain I); B, S324C (domain II); C, I526C (domain III); and D, L40C (domain I). • represents the fluorescence in buffer; ○ represents the fluorescence in BBMV before proteinase K treatment; and ▾ represents the fluorescence in BBMV after proteinase K treatment. y axis represents the relative intensity of fluorescence of the labeled mutant in buffer versus membranes and not absolute values of intensity.

FIGURE 6.

A, voltage clamp response of Cry1Ab (•) to those of domain I mutants as follows: L40C (□), D62C (•), S191C (Δ), L199C (○), and L215C (▾). V171C has been reported earlier (35). B, voltage clamp response of Cry1Aa (•) to those in domain II mutants as follows: S279C (○), S324C (▾), S364C (Δ), E460C (•), K489C (□), and I526C (♦). F371C and F371W have been reported earlier (34, 35). The arrow indicates the time at which toxin was added to the stabilized midguts.