Distinct biochemical and topological properties of the 31- and 27-kilodalton plasma membrane intrinsic protein subgroups from red beet

Abstract

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

Figure 1

Tissue distribution of BPM2 and BPM3 by RNA gel-blot analysis.

Figure 2

Suc-gradient centrifugation of a microsomal vesicle fraction. A microsomal membrane fraction (3.4 mg of protein) was centrifuged overnight at 80,000g on a continuous gradient of Suc (15%–45%, w/w). Top, Distribution of the PM marker callose synthase and Suc. Middle, Immunoblot probed with a monoclonal antibody to the 60-kD vacuolar H⁺-ATPase (1:1,000 dilution). Bottom, Immunoblot dually probed with affinity-purified polyclonal antibodies specific for the PMIP31 and PMIP27 species (1:2,000 dilution). Each lane contained 20 μL from each gradient fraction and was prepared for SDS-PAGE in 100 mm DTT.

Figure 3

Effects of Cys modification reagents. PM vesicles were isolated in the presence (+) and absence (−) of I-Ac (5 mm) and NEM (5 mm). Samples were treated with (+) or without (−) diamide (3 mm) before SDS-PAGE. Immunoblotting was performed using anti-PMIP27 (1:2500, A) and anti-PMIP31 (1:2000, B).

Figure 4

Different fragmentation patterns of PMIP31 and PMIP27. Limited proteolysis was conducted with PM vesicles treated with 0.01% digitonin. Proteases used were Pronase E (left) and trypsin (right). Membrane-bound proteolytic products were extracted, electrophoresed, and blotted with anti-PMIP27 and anti-PMIP31, as indicated.

Figure 5

Occlusion of tryptic site II in the presence of HgCl₂. A, PM vesicles were incubated with increasing levels of HgCl₂ and 0.01% digitonin before treatment with trypsin (40 μg mL⁻¹). B, Right-side-out vesicles were treated with trypsin in the absence (−) and presence (+) of 3 mm HgCl₂ and digitonin, as indicated. The immunoblots were probed with anti-PMIP27.

Figure 6

Topological model of a PMIP27. This model is based on a hydropathy plot (Kyte and Doolittle, 1982) of BPM3 (Qi et al., 1996) and the topological data provided here. The conserved domains located at surface loops C and E are shown. The locations of tryptic cleavage sites I and II are indicated. Conserved Cys residues and the two NPA domains of BPM3 are also shown.