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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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Extractions of cells expressing VLP54, VLP6 (a GFP fusion to the ER protein HO-2), and LBR-S (a GFP fusion to a truncated LBR). BHK cells were transiently transfected with expression constructs and fixed (no extraction) or extracted on ice with 1% TX-100 in PBS (1% TX-100), or 1% TX-100 in PBS supplemented with 350 mM NaCl (1% TX-100 + salt), or subjected to a nuclear matrix preparation (nuclear matrix) before fixation. Upper panels show GFP fluorescence and lower panels show corresponding phase-contrast images. All GFP images were taken at the same exposure and subsequently scaled identically. Bar, 20 μm.
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Figure 5: Extractions of cells expressing VLP54, VLP6 (a GFP fusion to the ER protein HO-2), and LBR-S (a GFP fusion to a truncated LBR). BHK cells were transiently transfected with expression constructs and fixed (no extraction) or extracted on ice with 1% TX-100 in PBS (1% TX-100), or 1% TX-100 in PBS supplemented with 350 mM NaCl (1% TX-100 + salt), or subjected to a nuclear matrix preparation (nuclear matrix) before fixation. Upper panels show GFP fluorescence and lower panels show corresponding phase-contrast images. All GFP images were taken at the same exposure and subsequently scaled identically. Bar, 20 μm.

Mentions: At low expression levels in transiently transfected cells, the predominant GFP pattern of VLP54 was nuclear rim. This pattern was also seen in a stable cell line we constructed (Fig. 4 c). At higher expression levels in transiently transfected cells, VLP54 was also present in the peripheral ER (see Fig. 5), suggesting that its targeting to the NE may be easily saturable. To determine whether endogenous nurim is also localized to the NE, we used the affinity-purified peptide antibodies for immunofluorescence. All four antibodies stained the NE but not peripheral ER (Fig. 4 d and not shown), confirming that endogenous nurim is localized like VLP54 to 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)

Extractions of cells expressing VLP54, VLP6 (a GFP fusion to the ER protein HO-2), and LBR-S (a GFP fusion to a truncated LBR). BHK cells were transiently transfected with expression constructs and fixed (no extraction) or extracted on ice with 1% TX-100 in PBS (1% TX-100), or 1% TX-100 in PBS supplemented with 350 mM NaCl (1% TX-100 + salt), or subjected to a nuclear matrix preparation (nuclear matrix) before fixation. Upper panels show GFP fluorescence and lower panels show corresponding phase-contrast images. All GFP images were taken at the same exposure and subsequently scaled identically. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Extractions of cells expressing VLP54, VLP6 (a GFP fusion to the ER protein HO-2), and LBR-S (a GFP fusion to a truncated LBR). BHK cells were transiently transfected with expression constructs and fixed (no extraction) or extracted on ice with 1% TX-100 in PBS (1% TX-100), or 1% TX-100 in PBS supplemented with 350 mM NaCl (1% TX-100 + salt), or subjected to a nuclear matrix preparation (nuclear matrix) before fixation. Upper panels show GFP fluorescence and lower panels show corresponding phase-contrast images. All GFP images were taken at the same exposure and subsequently scaled identically. Bar, 20 μm.
Mentions: At low expression levels in transiently transfected cells, the predominant GFP pattern of VLP54 was nuclear rim. This pattern was also seen in a stable cell line we constructed (Fig. 4 c). At higher expression levels in transiently transfected cells, VLP54 was also present in the peripheral ER (see Fig. 5), suggesting that its targeting to the NE may be easily saturable. To determine whether endogenous nurim is also localized to the NE, we used the affinity-purified peptide antibodies for immunofluorescence. All four antibodies stained the NE but not peripheral ER (Fig. 4 d and not shown), confirming that endogenous nurim is localized like VLP54 to 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