Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane

G H Miao; Z Hong; D P Verma

doi:10.1083/jcb.118.2.481

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Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane

Journal article

Open access

Peer reviewed

Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane

G H Miao, Z Hong and D P Verma

The Journal of cell biology, Vol.118(2), pp.481-490

07/1992

DOI: https://doi.org/10.1083/jcb.118.2.481

Handle:

https://hdl.handle.net/2376/117790

PMCID: PMC2290056

PMID: 1629242

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Abstract

Models, Structural

Phosphorylation

Protein Biosynthesis

Antibodies

Molecular Sequence Data

Soybeans - genetics

Polymerase Chain Reaction - methods

Cattle

Plant Proteins - chemistry

Base Sequence

Transcription, Genetic

Plant Proteins - metabolism

Eye Proteins - genetics

Membrane Glycoproteins

Oligopeptides - chemistry

Chromosome Deletion

Amino Acid Sequence

Promoter Regions, Genetic

Oligodeoxyribonucleotides

Membrane Proteins - genetics

Aquaporins

Oligopeptides - immunology

Phosphoproteins - genetics

Reticulocytes - metabolism

Sequence Homology, Nucleic Acid

DNA - genetics

Plant Proteins - genetics

Animals

Protein Conformation

Protein Processing, Post-Translational

Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules. It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence. A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter. In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide. The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion. Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated. Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A. Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids. In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane. This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane. Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not. Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.

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Title: Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane
Creators: G H Miao - Department of Molecular Genetics and Biotechnology Center, Ohio State University, Columbus 43210
Z Hong
D P Verma
Publication Details: The Journal of cell biology, Vol.118(2), pp.481-490
Academic Unit: Center for Reproductive Biology
Publisher: United States
Identifiers: 99900547908201842
Language: English
Resource Type: Journal article