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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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Localization and detergent sensitivity of GFP-fusions in chicken cells. (a) Chicken fibroblasts were transiently transfected with LBR-S and LAP2-S (GFP fusions to NE proteins), VLP25 (a GFP fusion to the ER protein Sec61β), or GFP-nurim. 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 in insets. GFP images were taken at the same exposure and scaled identically. (b) Chicken cells were transiently transfected with both CFP-lamin A and YFP-nurim and treated as in a. The cyan and yellow images of the same cells are shown in the top and bottom frames, respectively. All images were taken at the same exposure and subsequently scaled identically. Bars, 20 μm.
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Figure 8: Localization and detergent sensitivity of GFP-fusions in chicken cells. (a) Chicken fibroblasts were transiently transfected with LBR-S and LAP2-S (GFP fusions to NE proteins), VLP25 (a GFP fusion to the ER protein Sec61β), or GFP-nurim. 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 in insets. GFP images were taken at the same exposure and scaled identically. (b) Chicken cells were transiently transfected with both CFP-lamin A and YFP-nurim and treated as in a. The cyan and yellow images of the same cells are shown in the top and bottom frames, respectively. All images were taken at the same exposure and subsequently scaled identically. Bars, 20 μm.

Mentions: Cells were mounted in 90% glycerol/10% 0.2 M Tris, pH 7.4. For all experiments, except those shown in Fig. 7, Fig. 8 b, and 10, cells were viewed on an Axioplan II microscope (Carl Zeiss), equipped with an Orca 12-bit–cooled CCD camera (Hamamatsu Photonics). Images were captured and scaled using Image-Pro Plus 3.0 software (Media Cybernetics) with additions by Phase 3 Imaging Systems. A Zeiss 63× plan Apochromat oil immersion objective was used. For experiments in Fig. 7 and Fig. 8 b, a DeltaVision microscope system (Applied Precision Instruments) built around a Zeiss Axiovert microscope and with a PXL CCD camera (Photometrics Ltd.) was used with a Zeiss 100× plan Apochromat oil immersion objective (see Fig. 7) or a 63× plan Apochromat oil immersion objective (see Fig. 8 b). Filters for visualization of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were from Chroma Technology Corp. After acquisition of images with DeltaVision software they were exported to either NIH Image 1.62 (National Institutes of Health) for Fig. 7 or ImagePro Plus for Fig. 8 b, and scaling and overlaying were performed in these applications. Final preparation of all figures was done using Canvas 5.0 (Deneba Systems, Inc.).


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-fusions in chicken cells. (a) Chicken fibroblasts were transiently transfected with LBR-S and LAP2-S (GFP fusions to NE proteins), VLP25 (a GFP fusion to the ER protein Sec61β), or GFP-nurim. 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 in insets. GFP images were taken at the same exposure and scaled identically. (b) Chicken cells were transiently transfected with both CFP-lamin A and YFP-nurim and treated as in a. The cyan and yellow images of the same cells are shown in the top and bottom frames, respectively. All images were taken at the same exposure and subsequently scaled identically. Bars, 20 μm.
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Related In: Results  -  Collection

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Figure 8: Localization and detergent sensitivity of GFP-fusions in chicken cells. (a) Chicken fibroblasts were transiently transfected with LBR-S and LAP2-S (GFP fusions to NE proteins), VLP25 (a GFP fusion to the ER protein Sec61β), or GFP-nurim. 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 in insets. GFP images were taken at the same exposure and scaled identically. (b) Chicken cells were transiently transfected with both CFP-lamin A and YFP-nurim and treated as in a. The cyan and yellow images of the same cells are shown in the top and bottom frames, respectively. All images were taken at the same exposure and subsequently scaled identically. Bars, 20 μm.
Mentions: Cells were mounted in 90% glycerol/10% 0.2 M Tris, pH 7.4. For all experiments, except those shown in Fig. 7, Fig. 8 b, and 10, cells were viewed on an Axioplan II microscope (Carl Zeiss), equipped with an Orca 12-bit–cooled CCD camera (Hamamatsu Photonics). Images were captured and scaled using Image-Pro Plus 3.0 software (Media Cybernetics) with additions by Phase 3 Imaging Systems. A Zeiss 63× plan Apochromat oil immersion objective was used. For experiments in Fig. 7 and Fig. 8 b, a DeltaVision microscope system (Applied Precision Instruments) built around a Zeiss Axiovert microscope and with a PXL CCD camera (Photometrics Ltd.) was used with a Zeiss 100× plan Apochromat oil immersion objective (see Fig. 7) or a 63× plan Apochromat oil immersion objective (see Fig. 8 b). Filters for visualization of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were from Chroma Technology Corp. After acquisition of images with DeltaVision software they were exported to either NIH Image 1.62 (National Institutes of Health) for Fig. 7 or ImagePro Plus for Fig. 8 b, and scaling and overlaying were performed in these applications. Final preparation of all figures was done using Canvas 5.0 (Deneba Systems, Inc.).

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