Chromatographic and electrophoretic resolution of proteins and protein complexes from the larval midgut microvilli of Manduca sexta Yannick Pauchet a, * , Alexander Muck b , Ales ˇ Svatos ˇ b , David G. Heckel a a Entomology Department, Max Planck Institute for Chemical Ecology, Hans-Kno ¨ll-Str. 8, D-07745 Jena, Germany b Mass Spectrometry Research Group, Max Planck Institute for Chemical Ecology, Hans-Kno ¨ll-Str. 8, D-07745 Jena, Germany article info Article history: Received 10 March 2009 Received in revised form 11 May 2009 Accepted 13 May 2009 Keywords: Midgut proteome Microvilli Manduca sexta Protein complexes Aminopeptidase N Mass spectrometry abstract The microvillar proteome of Manduca sexta larval midguts was analyzed by subjecting brush border membrane vesicles (BBMV) to two different two-dimensional approaches: (i) Anion exchange chroma- tography followed by SDS-PAGE and (ii) Blue Native-PAGE followed by SDS-PAGE. The first technique was superior to conventional 2-D gel electrophoresis in resolving the most abundant proteins associated with the midgut microvilli. Twenty of them were successfully identified as digestive enzymes, binding targets of the insecticidal Cry1A toxins from Bacillus thuringiensis (Bt), and signal transduction proteins. A homolog of the chlorophyllide A binding protein from the silkworm and several aminopeptidases N represent the most abundant proteins associated with the BBMV. The second technique revealed protein oligomeric complexes associated with midgut microvilli in vivo. Two such complexes contained subunits of the vacuolar ATP synthase complex, and one was an oligomer of the chlorophyllide A binding protein. An additional complex consisted of homo- or hetero-tetramers of three different aminopeptidases N (APNs). As APNs are well-known binding partners of Cry1A toxins, their quaternary structure has implications for Bt toxin mode of action. Both techniques provide a useful complement to conventional 2-D gel electrophoresis in analyzing the complex proteome of the microvillar membrane fraction. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction In larvae of Lepidoptera, the midgut comprises most of the digestive tract making it the largest organ of the caterpillar, and performs most of the digestion and absorption of nutrients, generally at a high pH. The midgut is made up of a hollow tube of cells encircled by a basement matrix, lying within the hemocoel of the larva. The exterior of the basement matrix is bathed in hemo- lymph that carries nutrients to the rest of the body. Most of the inner surface of the basement matrix is covered by the basal ends of a single layer of cells, whose apical ends face the interior of the lumen (Terra and Ferreira, 2003). The two main cell types are columnar epithelial cells, with most of their apical surfaces elabo- rated into microvilli, and goblet cells, which possess a central cavity partly open to the lumen containing the proton vacuolar ATPase pump and associated Kþ/Hþ antiporter system responsible for maintaining a high Kþ gradient (Wieczorek et al., 2000) and driving amino acid uptake (Giordana et al., 1989). Stem cells lie between the basal regions of these two cell types and differentiate to replenish them. Preparations of columnar cell microvilli are termed brush border membrane vesicles (BBMV) and have been extensively studied in Lepidoptera not only because they contain the enzymes responsible for the last steps of digestion as well as transporters that bring nutrients into the cell (Terra, 2003), but also because they contain the binding partners of entomopathogenic toxins such as the Bacillus thuringiensis Cry toxins (Pigott and Ellar, 2007). To date, two different approaches for studying the larval midgut BBMV proteome of various Lepidoptera have been reported. The first is based on the outstanding separation capabilities of classical two- dimensional gel electrophoresis (2D-PAGE), with isoelectrofocus- ing in the first dimension followed by SDS-PAGE in the second dimension (Candas et al., 2003; Krishnamoorthy et al., 2007; McNall and Adang, 2003). The second approach is based on immunoscreening a midgut-specific cDNA library using antibodies raised against cytoskeleton-free microvillar preparations (Ferreira et al., 2007). Both methods have already allowed the character- ization of a substantial subset of the BBMV proteome of lepidop- teran larvae, however both methods have limitations. Severe solubility problems hamper the analysis of many classes of proteins by classical 2D-PAGE, especially membrane proteins which are presumably one of the main components of the BBMV. * Corresponding author. Centre for Ecology and Conservation, School of Biosci- ences, University of Exeter in Cornwall, Tremough campus, TR10 9EZ Penryn, Cornwall, United Kingdom. Tel.: þ441326371852; fax: þ441326253638. E-mail address: y.pauchet@exeter.ac.uk (Y. Pauchet). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2009.05.001 Insect Biochemistry and Molecular Biology 39 (2009) 467–474