Limits...
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.

Show MeSH
Examples of fluorescence patterns generated by GFP fusions to known NE proteins. BHK cells were transiently transfected with isolated clones (a) VLP4, a fusion to lamin A; (b) VLP23, a fusion to SUMO-1; and (c) VLP33, a fusion to the COOH-terminal half of emerin (starting at amino acid 103). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199743&req=5

Figure 3: Examples of fluorescence patterns generated by GFP fusions to known NE proteins. BHK cells were transiently transfected with isolated clones (a) VLP4, a fusion to lamin A; (b) VLP23, a fusion to SUMO-1; and (c) VLP33, a fusion to the COOH-terminal half of emerin (starting at amino acid 103). Bar, 20 μm.

Mentions: The NE was a pattern that could be clearly identified in the visual screen and we cloned a number of GFP fusions to known NE proteins. The most abundant class of NE proteins found was lamins. This is likely to reflect their abundance within the cell and the ease with which tagged lamins can be incorporated into the lamina. GFP–lamin fusions gave very clear, bright fluorescence at the nuclear rim and some internal nuclear structures (Fig. 3 a) that are likely to be invaginations of the NE 14. After identifying five independent clones of lamin A or C (which differ only in a splice variation at their COOH terminus) in the first 100 pools screened, we introduced a secondary screen to eliminate pools that exhibited NE fluorescence and expressed human lamin A or C. After this modification, we cloned only lamin B.


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)

Examples of fluorescence patterns generated by GFP fusions to known NE proteins. BHK cells were transiently transfected with isolated clones (a) VLP4, a fusion to lamin A; (b) VLP23, a fusion to SUMO-1; and (c) VLP33, a fusion to the COOH-terminal half of emerin (starting at amino acid 103). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199743&req=5

Figure 3: Examples of fluorescence patterns generated by GFP fusions to known NE proteins. BHK cells were transiently transfected with isolated clones (a) VLP4, a fusion to lamin A; (b) VLP23, a fusion to SUMO-1; and (c) VLP33, a fusion to the COOH-terminal half of emerin (starting at amino acid 103). Bar, 20 μm.
Mentions: The NE was a pattern that could be clearly identified in the visual screen and we cloned a number of GFP fusions to known NE proteins. The most abundant class of NE proteins found was lamins. This is likely to reflect their abundance within the cell and the ease with which tagged lamins can be incorporated into the lamina. GFP–lamin fusions gave very clear, bright fluorescence at the nuclear rim and some internal nuclear structures (Fig. 3 a) that are likely to be invaginations of the NE 14. After identifying five independent clones of lamin A or C (which differ only in a splice variation at their COOH terminus) in the first 100 pools screened, we introduced a secondary screen to eliminate pools that exhibited NE fluorescence and expressed human lamin A or C. After this modification, we cloned only lamin B.

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