Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis delta-endotoxin

Abstract

Bacillus thuringiensis delta-endotoxins insert into the brush border membranes of insect larval cells to form ion channels. A possible interaction of these toxins with a cytoplasmic component was examined by preloading vesicles from insect larval cells with protease K followed by incubation with toxin. There was no evidence for toxin antigens smaller than the intact toxin in extracts of solubilized vesicles, nor was there an effect of the inclusion of protease K on either of two functional properties, the formation of toxin aggregates or of ion pores. These toxins, physically and functionally, appear to be confined to the membrane.

FIG. 1

SDS-PAGE profile of BSA incubated with intact or lysed BBMV. Lane 1, standards of 205, 116, 97, 66, and 45 kDa (from top to bottom); lane 2, 2 μg of BSA; lanes 3 and 4, BSA incubated for 60 (lane 3) and 30 (lane 4) min with solubilized BBMV; lanes 5 and 6, BSA incubated with intact BBMV for 60 (lane 5) or 30 (lane 6) min; lanes 7 and 8, BSA incubated with solubilized BBMV containing protease K for 60 (lane 7) or 30 (lane 8) min; lanes 9 and 10, BSA incubated with intact BBMV containing protease K for 60 (lane 9) or 30 (lane 10) min.

FIG. 2

Immunoblot with aminopeptidase N antibody of solubilized M. sexta BBMV produced in the absence (lane 1) or presence (lane 2) of protease K. Lane 3, standards of 205, 116, 97, and 66 kDa (from top to bottom).

FIG. 3

Immunoblot of extracts of BBMV which had been preincubated with Cry1Ac1 toxin. Lane 1, BBMV (30 μg of protein) incubated with 100 ng of toxin; lane 2, BBMV (30 μg of protein) formed in the presence of protease K incubated with 100 ng of toxin; lane 3, 50 ng of Cry1Ac1 toxin. STD, standards of 66, 97, 116, and 205 kDa (with an extra band of >205 kDa), from bottom to top.

FIG. 4

Normalized light scattering of vesicles formed in the absence or presence of protease K (PK). The averages of three measurements taken over 50 s are plotted (standard deviations were 2 to 4%).