Abstract
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.
Highlights
Joined together at the nuclear pore complex, the center of which forms a channel controlling selective transport of molecules between the cytoplasm and the nucleoplasm [3, 4]
Nuclear membrane proteins that have been characterized to date contain extended N- or C-terminal domains that are thought to anchor the proteins to the inner nuclear membrane (INM) by interactions with various nuclear components, including the lamina and chromatin-associated proteins [2]
In an attempt to further characterize nurim membrane topology and compartmentalization at the INM, we fused nurim to green fluorescent protein (GFP) and examined the localization by imaging and biochemical fractionation. (The examination of GFP fusion proteins appeared suitable since nurim was first identified as an INM protein in a screening approach of a GFP fusion library.) We constructed three different nurim-GFP constructs (Fig. 1c)
Summary
Joined together at the nuclear pore complex, the center of which forms a channel controlling selective transport of molecules between the cytoplasm and the nucleoplasm [3, 4]. We examine INM association and the topological model of nurim in the membrane using green fluorescent protein (GFP) fusion derivatives of the protein in localization studies, biochemical extraction studies, and endoproteinase cleavage analysis.
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