title: 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.
authors.affiliation: Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901-8520, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Barone
authors.forename: H H
authors.initials: HH
authors.lastname: Mu
authors.forename: C J
authors.initials: CJ
authors.lastname: Shih
authors.forename: K B
authors.initials: KB
authors.lastname: Kashlan
authors.forename: B P
authors.initials: BP
authors.lastname: Wasserman
mesh_terms: D000595:Amino Acid Sequence; D020346:Aquaporins; D019663:Chenopodiaceae / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D017343:Genes, Plant; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D013439:Sulfhydryl Reagents; D014018:Tissue Distribution
chemical_list: D020346:Aquaporins; D007473:Ion Channels; D010446:Peptide Fragments; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D013439:Sulfhydryl Reagents; C110065:major intrinsic protein, plant