DISTINCT BIOCHEMICAL AND TOPOLOGICAL PROPERTIES OF THE 31-AND 27-KILODALTON PLASMA-MEMBRANE INTRINSIC PROTEIN SUBGROUPS FROM RED BEET

Plasma membrane Vesicles from red beet (Beta vulgaris L.) storage tiss ue 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 t heir localization and biochemical properties. Both plasma membrane int rinsic protein (PMIP) subgroups partitioned identically in sucrose gra dients; however, each exhibited distinct properties when probed for mu ltimer formation, and by limited proteolysis. The tendency of each PMI P 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 sulthydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contr ast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulf ide-linked heterodimers between the PMIP31 and PMIP27 subgroups were n ot detected. Based on vectorial proteolysis of right-side-out Vesicles with trypsin and hydropathy analysis of the predicted amino acid sequ ence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identi fied from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Alt hough the PMIP31 and PMIP27 species both contain integral proteins tha t appear to occur within a single vesicle population, these results de monstrate that each PMIP subgroup responds differently to perturbation s of the membrane.