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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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Examples of fluorescence patterns generated by expression of individual clones isolated in the visual screen. Individual clones were transiently transfected into BHK cells. (a) A mitochondrial pattern is shown by VLP32, a GFP fusion to ADP/ATP translocase; (b) an ER pattern by VLP16, a fusion to the signal peptidase 25 kD subunit; (c) a cytoskeletal pattern by VLP11, a fusion to β-actin; (d) a chromatin pattern by VLP51, a fusion to histone H1; (e) a nucleolar pattern by VLP56, a fusion to ribosomal protein L27; and (f) a centrosomal pattern by VLP31, a fusion to the ATCase domain of CAD. Bar, 20 μm.
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Figure 2: Examples of fluorescence patterns generated by expression of individual clones isolated in the visual screen. Individual clones were transiently transfected into BHK cells. (a) A mitochondrial pattern is shown by VLP32, a GFP fusion to ADP/ATP translocase; (b) an ER pattern by VLP16, a fusion to the signal peptidase 25 kD subunit; (c) a cytoskeletal pattern by VLP11, a fusion to β-actin; (d) a chromatin pattern by VLP51, a fusion to histone H1; (e) a nucleolar pattern by VLP56, a fusion to ribosomal protein L27; and (f) a centrosomal pattern by VLP31, a fusion to the ATCase domain of CAD. Bar, 20 μm.

Mentions: The group of 27 clones targeted to single, defined subcellular compartments included 25 sequences of known proteins (Table ). The fluorescence patterns of most of these fusion proteins were consistent with their localization to the same compartment as the endogenous protein (exceptions noted in Table ). In most cases the clones encoding correctly localized fusions contained the entire coding sequence of the endogenous proteins. Examples of the patterns obtained with isolated clones are shown in Fig. 2. The correct localization of GFP fusions to known proteins confirmed that this visual screen yields meaningful results when a pattern of interest can be clearly identified.


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 expression of individual clones isolated in the visual screen. Individual clones were transiently transfected into BHK cells. (a) A mitochondrial pattern is shown by VLP32, a GFP fusion to ADP/ATP translocase; (b) an ER pattern by VLP16, a fusion to the signal peptidase 25 kD subunit; (c) a cytoskeletal pattern by VLP11, a fusion to β-actin; (d) a chromatin pattern by VLP51, a fusion to histone H1; (e) a nucleolar pattern by VLP56, a fusion to ribosomal protein L27; and (f) a centrosomal pattern by VLP31, a fusion to the ATCase domain of CAD. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Examples of fluorescence patterns generated by expression of individual clones isolated in the visual screen. Individual clones were transiently transfected into BHK cells. (a) A mitochondrial pattern is shown by VLP32, a GFP fusion to ADP/ATP translocase; (b) an ER pattern by VLP16, a fusion to the signal peptidase 25 kD subunit; (c) a cytoskeletal pattern by VLP11, a fusion to β-actin; (d) a chromatin pattern by VLP51, a fusion to histone H1; (e) a nucleolar pattern by VLP56, a fusion to ribosomal protein L27; and (f) a centrosomal pattern by VLP31, a fusion to the ATCase domain of CAD. Bar, 20 μm.
Mentions: The group of 27 clones targeted to single, defined subcellular compartments included 25 sequences of known proteins (Table ). The fluorescence patterns of most of these fusion proteins were consistent with their localization to the same compartment as the endogenous protein (exceptions noted in Table ). In most cases the clones encoding correctly localized fusions contained the entire coding sequence of the endogenous proteins. Examples of the patterns obtained with isolated clones are shown in Fig. 2. The correct localization of GFP fusions to known proteins confirmed that this visual screen yields meaningful results when a pattern of interest can be clearly identified.

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