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A visual screen of a GFP-fusion library identifies a new type of nuclear envelope membrane protein.

Rolls MM, Stein PA, Taylor SS, Ha E, McKeon F, Rapoport TA - J. Cell Biol. (1999)

Bottom Line: This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER.Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus.Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

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Related in: MedlinePlus

Localization and detergent sensitivity of GFP-nurim deletions and point mutants. BHK cells were transiently transfected with nurim-tagged with GFP at the COOH terminus rather than the NH2 terminus (54C) or loop deletions (Δ1 and Δ2), truncation (T16), or point mutations (D66L, R98L, and R217L); diagrams of the mutants are included to the right with shading representing predicted transmembrane domains. Cells were either fixed immediately (no extraction) or extracted with 1% TX-100 before fixation (1% TX-100). GFP fluorescence is shown in large images and Hoechst staining to show nuclei in insets. All GFP images were taken at the same exposure and scaled identically. Bar, 20 μm.
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Figure 9: Localization and detergent sensitivity of GFP-nurim deletions and point mutants. BHK cells were transiently transfected with nurim-tagged with GFP at the COOH terminus rather than the NH2 terminus (54C) or loop deletions (Δ1 and Δ2), truncation (T16), or point mutations (D66L, R98L, and R217L); diagrams of the mutants are included to the right with shading representing predicted transmembrane domains. Cells were either fixed immediately (no extraction) or extracted with 1% TX-100 before fixation (1% TX-100). GFP fluorescence is shown in large images and Hoechst staining to show nuclei in insets. All GFP images were taken at the same exposure and scaled identically. Bar, 20 μm.

Mentions: To determine whether a nucleoplasmic region of nurim could function independently to target the protein to the NE, we examined the targeting of GFP-nurim mutants. Unlike the known members of the lamin-associated class, nurim does not have a long NH2-terminal extension before its first transmembrane domain that could contain an NE targeting domain. However, it does have several short regions that could extend into the nucleus. We made deletions in these regions: the two longest loops between transmembrane domains, deletions Δ1 and Δ2, and the tail after the last transmembrane domain. Removal of the last 16 amino acids of nurim, mutation T16, had no effect on targeting to the NE or detergent inextractibility (Fig. 9). On the other hand, mutants Δ1 and Δ2 were no longer concentrated in the NE, but were distributed throughout the ER, and were completely extracted by 1% TX-100 (Fig. 9). The two loops in which we made deletions are predicted to be on opposite sides of the membrane. Obtaining similar results with both deletions does not support the idea that one domain would reach into the nucleus and anchor the protein in the NE.


A visual screen of a GFP-fusion library identifies a new type of nuclear envelope membrane protein.

Rolls MM, Stein PA, Taylor SS, Ha E, McKeon F, Rapoport TA - J. Cell Biol. (1999)

Localization and detergent sensitivity of GFP-nurim deletions and point mutants. BHK cells were transiently transfected with nurim-tagged with GFP at the COOH terminus rather than the NH2 terminus (54C) or loop deletions (Δ1 and Δ2), truncation (T16), or point mutations (D66L, R98L, and R217L); diagrams of the mutants are included to the right with shading representing predicted transmembrane domains. Cells were either fixed immediately (no extraction) or extracted with 1% TX-100 before fixation (1% TX-100). GFP fluorescence is shown in large images and Hoechst staining to show nuclei in insets. All GFP images were taken at the same exposure and scaled identically. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199743&req=5

Figure 9: Localization and detergent sensitivity of GFP-nurim deletions and point mutants. BHK cells were transiently transfected with nurim-tagged with GFP at the COOH terminus rather than the NH2 terminus (54C) or loop deletions (Δ1 and Δ2), truncation (T16), or point mutations (D66L, R98L, and R217L); diagrams of the mutants are included to the right with shading representing predicted transmembrane domains. Cells were either fixed immediately (no extraction) or extracted with 1% TX-100 before fixation (1% TX-100). GFP fluorescence is shown in large images and Hoechst staining to show nuclei in insets. All GFP images were taken at the same exposure and scaled identically. Bar, 20 μm.
Mentions: To determine whether a nucleoplasmic region of nurim could function independently to target the protein to the NE, we examined the targeting of GFP-nurim mutants. Unlike the known members of the lamin-associated class, nurim does not have a long NH2-terminal extension before its first transmembrane domain that could contain an NE targeting domain. However, it does have several short regions that could extend into the nucleus. We made deletions in these regions: the two longest loops between transmembrane domains, deletions Δ1 and Δ2, and the tail after the last transmembrane domain. Removal of the last 16 amino acids of nurim, mutation T16, had no effect on targeting to the NE or detergent inextractibility (Fig. 9). On the other hand, mutants Δ1 and Δ2 were no longer concentrated in the NE, but were distributed throughout the ER, and were completely extracted by 1% TX-100 (Fig. 9). The two loops in which we made deletions are predicted to be on opposite sides of the membrane. Obtaining similar results with both deletions does not support the idea that one domain would reach into the nucleus and anchor the protein in the NE.

Bottom Line: This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER.Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus.Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

Show MeSH
Related in: MedlinePlus