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 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.
Barone
L M
LM
Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901-8520, USA.
Mu
H H
HH
Shih
C J
CJ
Kashlan
K B
KB
Wasserman
B P
BP
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
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