An essential 45 kDa yeast transmembrane protein reacts with anti-nuclear pore antibodies: purification of the protein, immunolocalization and cloning of the gene.
A yeast membrane protein was isolated by its binding to tRNA Sepharose column. The 45 kDa protein shares characteristics with rat liver nuclear pore proteins in having reactivity with a monoclonal antibody (RL1) raised against rat liver nuclear pore proteins and by the binding of wheat germ agglutinin (WGA), indicating the presence of N-acetylglucosamine (GlcNAc) moieties. Immunofluorescence microscopy and cell fractionation experiments indicate that the protein is located in the nuclear envelope and the endoplasmic reticulum of the cell. The gene for the 45 kDa protein was cloned using degenerate oligonucleotides derived from the N-terminal protein sequence and confirmed by internal peptide sequences. The gene was named WBP1. The protein coding sequence of the WBP1 gene reveals an ER entry signal peptide and a C-terminal membrane spanning domain. Topological studies indicate that the C-terminus of the protein is located in the cytoplasm. The cytoplasmic tail of the protein contains the K-K-X-X signal known to be sufficient for retention of transmembrane proteins in higher eukaryotic cells. Gene disruption experiments show that the 45 kDa protein is essential for the vegetative life cycle of the yeast cell.
te Heesen
S
S
Institut für Molekularbiologie I, Universität, Zürich/Switzerland.
Rauhut
R
R
Aebersold
R
R
Abelson
J
J
Aebi
M
M
Clark
M W
MW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000737:chemistry* / Q000276:immunology; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000737:chemistry / Q000276:immunology; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009687:Nuclear Proteins; D014909:Wheat Germ Agglutinins