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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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Association of VLP54 and endogenous nurim with extracted nuclear pellets. (a) BHK cells stably expressing either VLP54 or a cyan version of VLP6 (HO-2) and yellow version of VLP25 (Sec61β) were treated with PBS, PBS with 1% TX-100, or PBS with 1% TX-100 and high salt. Nuclei were pelleted, washed, and analyzed by SDS-PAGE followed by immunoblotting with an anti-GFP antibody. Four times the amount of extracted nuclei compared with unextracted nuclei (1 × 106) were analyzed. (b) HeLa cell nuclei were treated as in a, and analyzed by immunoblotting with affinity-purified antinurim antibody 254. In this experiment supernatants were also collected and the amount from equivalent numbers of cells (3 × 105) were loaded in all lanes.
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Figure 6: Association of VLP54 and endogenous nurim with extracted nuclear pellets. (a) BHK cells stably expressing either VLP54 or a cyan version of VLP6 (HO-2) and yellow version of VLP25 (Sec61β) were treated with PBS, PBS with 1% TX-100, or PBS with 1% TX-100 and high salt. Nuclei were pelleted, washed, and analyzed by SDS-PAGE followed by immunoblotting with an anti-GFP antibody. Four times the amount of extracted nuclei compared with unextracted nuclei (1 × 106) were analyzed. (b) HeLa cell nuclei were treated as in a, and analyzed by immunoblotting with affinity-purified antinurim antibody 254. In this experiment supernatants were also collected and the amount from equivalent numbers of cells (3 × 105) were loaded in all lanes.

Mentions: Cells were removed from 10-cm tissue culture plates with PBS plus 5 mM EDTA, pelleted at 1,000 g for 5 min and washed in cold PBS+. For some experiments (see Fig. 6 a), the cells were divided directly into three samples, pelleted, and treated with one of three extraction conditions: PBS+ alone (control), PBS+ with 1% TX-100, or PBS+ with 1% TX and 350 mM NaCl, each supplemented with 1 mM DTT and protease inhibitor cocktail. Samples were incubated for 30 min on ice. The insoluble material was pelleted at 3,500 g for 10 min, washed with PBS+, and repelleted. In other experiments (see Fig. 4 b and 6 b) total membrane fractions (BHK cells and BHK cells overexpressing nurim coding sequence) or nuclei (HeLa cells and Vero cells) were first isolated by hypotonic lysis. Cells were incubated in 10 vol of cold hypotonic lysis buffer (HLB: 10 mM Tris-Cl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail) until swollen and lysed by passage through a ball bearing homogenizer. Extent of lysis was monitored by phase-contrast microscopy. Total membranes were collected by centrifugation at 25,000 g for 10 min, washed in cold isotonic buffer (ILB: 10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail), and resuspended in nuclei resuspension buffer (NRB: 15 mM Hepes, pH 7.4, 80 mM KCl, 15 mM NaCl, 250 mM sucrose, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail). Nuclei were collected by centrifugation at 3,500 g for 5 min, washed, and repelleted twice in NRB. Aliquots from these samples were either directly solubilized in SDS-PAGE sample buffer (see Fig. 4 b) or extracted as described above (see Fig. 6 b). To analyze supernatants, proteins were precipitated with TCA (15% final), pelleted at 25,000 g for 10 min, and pellets were washed twice with acetone. Final pellets were solubilized in SDS-PAGE sample buffer at 65°C for at least 30 min. For immunoblot analysis, proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed either with rabbit polyclonal anti-GFP antibodies (provided by P. Silver, Dana-Farber Cancer Institute, Boston, MA) at a dilution of 1:3,000 or with rabbit polyclonal antinurim antibodies at a dilution of 1:4,000 in TBS/0.1% Tween 20 with 5% dry milk and proteins were detected by chemiluminescence (Renaissance; NEN Life Science Products).


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)

Association of VLP54 and endogenous nurim with extracted nuclear pellets. (a) BHK cells stably expressing either VLP54 or a cyan version of VLP6 (HO-2) and yellow version of VLP25 (Sec61β) were treated with PBS, PBS with 1% TX-100, or PBS with 1% TX-100 and high salt. Nuclei were pelleted, washed, and analyzed by SDS-PAGE followed by immunoblotting with an anti-GFP antibody. Four times the amount of extracted nuclei compared with unextracted nuclei (1 × 106) were analyzed. (b) HeLa cell nuclei were treated as in a, and analyzed by immunoblotting with affinity-purified antinurim antibody 254. In this experiment supernatants were also collected and the amount from equivalent numbers of cells (3 × 105) were loaded in all lanes.
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Figure 6: Association of VLP54 and endogenous nurim with extracted nuclear pellets. (a) BHK cells stably expressing either VLP54 or a cyan version of VLP6 (HO-2) and yellow version of VLP25 (Sec61β) were treated with PBS, PBS with 1% TX-100, or PBS with 1% TX-100 and high salt. Nuclei were pelleted, washed, and analyzed by SDS-PAGE followed by immunoblotting with an anti-GFP antibody. Four times the amount of extracted nuclei compared with unextracted nuclei (1 × 106) were analyzed. (b) HeLa cell nuclei were treated as in a, and analyzed by immunoblotting with affinity-purified antinurim antibody 254. In this experiment supernatants were also collected and the amount from equivalent numbers of cells (3 × 105) were loaded in all lanes.
Mentions: Cells were removed from 10-cm tissue culture plates with PBS plus 5 mM EDTA, pelleted at 1,000 g for 5 min and washed in cold PBS+. For some experiments (see Fig. 6 a), the cells were divided directly into three samples, pelleted, and treated with one of three extraction conditions: PBS+ alone (control), PBS+ with 1% TX-100, or PBS+ with 1% TX and 350 mM NaCl, each supplemented with 1 mM DTT and protease inhibitor cocktail. Samples were incubated for 30 min on ice. The insoluble material was pelleted at 3,500 g for 10 min, washed with PBS+, and repelleted. In other experiments (see Fig. 4 b and 6 b) total membrane fractions (BHK cells and BHK cells overexpressing nurim coding sequence) or nuclei (HeLa cells and Vero cells) were first isolated by hypotonic lysis. Cells were incubated in 10 vol of cold hypotonic lysis buffer (HLB: 10 mM Tris-Cl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail) until swollen and lysed by passage through a ball bearing homogenizer. Extent of lysis was monitored by phase-contrast microscopy. Total membranes were collected by centrifugation at 25,000 g for 10 min, washed in cold isotonic buffer (ILB: 10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail), and resuspended in nuclei resuspension buffer (NRB: 15 mM Hepes, pH 7.4, 80 mM KCl, 15 mM NaCl, 250 mM sucrose, 1.5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail). Nuclei were collected by centrifugation at 3,500 g for 5 min, washed, and repelleted twice in NRB. Aliquots from these samples were either directly solubilized in SDS-PAGE sample buffer (see Fig. 4 b) or extracted as described above (see Fig. 6 b). To analyze supernatants, proteins were precipitated with TCA (15% final), pelleted at 25,000 g for 10 min, and pellets were washed twice with acetone. Final pellets were solubilized in SDS-PAGE sample buffer at 65°C for at least 30 min. For immunoblot analysis, proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed either with rabbit polyclonal anti-GFP antibodies (provided by P. Silver, Dana-Farber Cancer Institute, Boston, MA) at a dilution of 1:3,000 or with rabbit polyclonal antinurim antibodies at a dilution of 1:4,000 in TBS/0.1% Tween 20 with 5% dry milk and proteins were detected by chemiluminescence (Renaissance; NEN Life Science Products).

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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