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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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FRAP of GFP-fusions to NE and ER proteins. (a) BHK cells transiently transfected with GFP-nurim or point mutant D66L were imaged with a confocal microscope. A portion of the cell (box 4) was subjected to photobleaching and fluorescence recovery monitored by imaging every 11s for 220 s, and then every minute for 5 min. Examples of images of cells at various times after recovery are shown. (b) FRAP experiments were performed as in a for GFP-nurim, D66L, VLP25 (a GFP fusion to Sec61β), YFP-emerin, LAP2-S, and LBR-S. The results from three bleached cells were quantitated and combined and the SD indicated with a bar (some of the SDs for early time points are not shown in the plots, but were similar to those shown). For quantitation, the total pixel intensity in a region of the cell that included the NE (a, box 1) was calculated. The intensity of an equivalent sized background area (box 3) was subtracted and, finally, the images were normalized for brightness using an unbleached region of the cell (box 2). Bar, 10 μm.
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Figure 10: FRAP of GFP-fusions to NE and ER proteins. (a) BHK cells transiently transfected with GFP-nurim or point mutant D66L were imaged with a confocal microscope. A portion of the cell (box 4) was subjected to photobleaching and fluorescence recovery monitored by imaging every 11s for 220 s, and then every minute for 5 min. Examples of images of cells at various times after recovery are shown. (b) FRAP experiments were performed as in a for GFP-nurim, D66L, VLP25 (a GFP fusion to Sec61β), YFP-emerin, LAP2-S, and LBR-S. The results from three bleached cells were quantitated and combined and the SD indicated with a bar (some of the SDs for early time points are not shown in the plots, but were similar to those shown). For quantitation, the total pixel intensity in a region of the cell that included the NE (a, box 1) was calculated. The intensity of an equivalent sized background area (box 3) was subtracted and, finally, the images were normalized for brightness using an unbleached region of the cell (box 2). Bar, 10 μm.

Mentions: When we bleached part of the NE of a cell expressing low levels of GFP-nurim, we observed only limited recovery over a 9-min observation time (Fig. 10 a). On the other hand, the NE of cells expressing mutant D66L regained fluorescence during this period (Fig. 10 a). For comparison, we monitored the behavior of VLP25 (a GFP fusion to an ER protein), YFP-emerin, LAP2-S, and LBR-S, and quantitated the percent fluorescence recovery to the NE during the observation period. Like the fluorescence of D66L, that of ER protein VLP25 recovered rapidly to the bleached area (Fig. 10 b). On the other hand, fluorescence of the NE proteins recovered slowly with kinetics similar to those observed for GFP-nurim (Fig. 10 b). This result corroborated the tight association of nurim with components of the nucleus, indicated by its inextractability from the nuclear periphery with detergent and high salt. It also confirmed that mutation of a charged residue predicted to be in the second transmembrane domain disrupts targeting of GFP-nurim to the NE and results in a protein that behaves like a freely diffusible ER component.


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)

FRAP of GFP-fusions to NE and ER proteins. (a) BHK cells transiently transfected with GFP-nurim or point mutant D66L were imaged with a confocal microscope. A portion of the cell (box 4) was subjected to photobleaching and fluorescence recovery monitored by imaging every 11s for 220 s, and then every minute for 5 min. Examples of images of cells at various times after recovery are shown. (b) FRAP experiments were performed as in a for GFP-nurim, D66L, VLP25 (a GFP fusion to Sec61β), YFP-emerin, LAP2-S, and LBR-S. The results from three bleached cells were quantitated and combined and the SD indicated with a bar (some of the SDs for early time points are not shown in the plots, but were similar to those shown). For quantitation, the total pixel intensity in a region of the cell that included the NE (a, box 1) was calculated. The intensity of an equivalent sized background area (box 3) was subtracted and, finally, the images were normalized for brightness using an unbleached region of the cell (box 2). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 10: FRAP of GFP-fusions to NE and ER proteins. (a) BHK cells transiently transfected with GFP-nurim or point mutant D66L were imaged with a confocal microscope. A portion of the cell (box 4) was subjected to photobleaching and fluorescence recovery monitored by imaging every 11s for 220 s, and then every minute for 5 min. Examples of images of cells at various times after recovery are shown. (b) FRAP experiments were performed as in a for GFP-nurim, D66L, VLP25 (a GFP fusion to Sec61β), YFP-emerin, LAP2-S, and LBR-S. The results from three bleached cells were quantitated and combined and the SD indicated with a bar (some of the SDs for early time points are not shown in the plots, but were similar to those shown). For quantitation, the total pixel intensity in a region of the cell that included the NE (a, box 1) was calculated. The intensity of an equivalent sized background area (box 3) was subtracted and, finally, the images were normalized for brightness using an unbleached region of the cell (box 2). Bar, 10 μm.
Mentions: When we bleached part of the NE of a cell expressing low levels of GFP-nurim, we observed only limited recovery over a 9-min observation time (Fig. 10 a). On the other hand, the NE of cells expressing mutant D66L regained fluorescence during this period (Fig. 10 a). For comparison, we monitored the behavior of VLP25 (a GFP fusion to an ER protein), YFP-emerin, LAP2-S, and LBR-S, and quantitated the percent fluorescence recovery to the NE during the observation period. Like the fluorescence of D66L, that of ER protein VLP25 recovered rapidly to the bleached area (Fig. 10 b). On the other hand, fluorescence of the NE proteins recovered slowly with kinetics similar to those observed for GFP-nurim (Fig. 10 b). This result corroborated the tight association of nurim with components of the nucleus, indicated by its inextractability from the nuclear periphery with detergent and high salt. It also confirmed that mutation of a charged residue predicted to be in the second transmembrane domain disrupts targeting of GFP-nurim to the NE and results in a protein that behaves like a freely diffusible ER component.

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