Analysis of the localization and topology of nurim, a polytopic protein tightly associated with the inner nuclear membrane.
Nurim is an inner nuclear membrane (INM) protein that was first isolated in a visual screen for nuclear envelope-localizing proteins. Nurim lacks an N-terminal domain characteristic of other INM proteins examined to date and may represent a class of proteins that localize to the INM by a distinct mechanism. To further characterize this protein, we constructed nurim-green fluorescent protein fusions and analyzed aspects of localization, biochemistry, and membrane topology. Results from immunoprobing and protease protection assays together with other analyses indicate that nurim (total length of 262 residues) is a six transmembrane-spanning protein and contains a hairpin turn in its C-terminal transmembrane domain, resulting in the N and C termini residing on the same side of the membrane. A loop region between the fourth and fifth transmembrane domains is exposed toward the nucleoplasm and contains a region accessible for site-specific endoproteinase cleavage. In biochemical fractionation, nurim remained extremely tightly bound to nuclear fractions and was released in significant quantities only in the presence of 4 m urea. Under conditions in which nuclear lamins were completely extracted, a significant population of nurim remained resistant to solubilization. This tight binding requires the C-terminal region of the protein. DNase treatment only marginally influenced its retention characteristics in nuclei. Results from consideration of sequence alignments and identification of specific topological features of nurim indicate that it may possess enzymic function. These results are discussed with reference to the retention mechanism and possible nuclear function of nurim.
Hofemeister
Helmut
H
Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom.
O'Hare
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018076:DNA, Complementary; D003902:Detergents; D008565:Membrane Proteins; D009687:Nuclear Proteins; C120443:nurim; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases