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

Related in: MedlinePlus

Overview of the visual screen. (a) Construction of the expression library. Human cDNAs from an osteosarcoma cell line were inserted into a mammalian expression vector derived from pcDNA3. The vector was modified by the addition of part of the 5′ untranslated region of lamin A (lamin 5′ utr), as well as the coding sequences of a myc tag, a GFP variant (S65T and V163A), and a flexible linker (GGGLDPRVR). The library was initially generated as 20 master pools of 25,000 clones each. (b) Screening the expression library. Starting pools were generated by subdividing each master pool into 20 pools of 2,000 clones. These pools were screened for localized proteins by transfecting each pool into ∼2 × 104 cells grown on a coverslip. The transfection efficiency varied from 20–50% meaning that each clone in the pool was expressed in several cells. Transfected pools were screened visually by epifluorescence microscopy. Pools that gave rise to interesting patterns were retrieved from glycerol stocks and further subdivided and screened until single clones were isolated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199743&req=5

Figure 1: Overview of the visual screen. (a) Construction of the expression library. Human cDNAs from an osteosarcoma cell line were inserted into a mammalian expression vector derived from pcDNA3. The vector was modified by the addition of part of the 5′ untranslated region of lamin A (lamin 5′ utr), as well as the coding sequences of a myc tag, a GFP variant (S65T and V163A), and a flexible linker (GGGLDPRVR). The library was initially generated as 20 master pools of 25,000 clones each. (b) Screening the expression library. Starting pools were generated by subdividing each master pool into 20 pools of 2,000 clones. These pools were screened for localized proteins by transfecting each pool into ∼2 × 104 cells grown on a coverslip. The transfection efficiency varied from 20–50% meaning that each clone in the pool was expressed in several cells. Transfected pools were screened visually by epifluorescence microscopy. Pools that gave rise to interesting patterns were retrieved from glycerol stocks and further subdivided and screened until single clones were isolated.

Mentions: To identify proteins localized to different subcellular compartments, a library was constructed with poly(A)-primed cDNA prepared from a human osteosarcoma cell line. The library vector allowed expression of cDNAs in tissue culture cells as COOH-terminal polypeptide fusions to GFP (Fig. 1 a). While GFP is larger than most antibody epitope tags, it forms a tight structure and has been fused to many proteins without disrupting them. Its advantage for a large-scale screen is that it can be directly visualized in transfected cells without the manipulations required for epitope tags.


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)

Overview of the visual screen. (a) Construction of the expression library. Human cDNAs from an osteosarcoma cell line were inserted into a mammalian expression vector derived from pcDNA3. The vector was modified by the addition of part of the 5′ untranslated region of lamin A (lamin 5′ utr), as well as the coding sequences of a myc tag, a GFP variant (S65T and V163A), and a flexible linker (GGGLDPRVR). The library was initially generated as 20 master pools of 25,000 clones each. (b) Screening the expression library. Starting pools were generated by subdividing each master pool into 20 pools of 2,000 clones. These pools were screened for localized proteins by transfecting each pool into ∼2 × 104 cells grown on a coverslip. The transfection efficiency varied from 20–50% meaning that each clone in the pool was expressed in several cells. Transfected pools were screened visually by epifluorescence microscopy. Pools that gave rise to interesting patterns were retrieved from glycerol stocks and further subdivided and screened until single clones were isolated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Overview of the visual screen. (a) Construction of the expression library. Human cDNAs from an osteosarcoma cell line were inserted into a mammalian expression vector derived from pcDNA3. The vector was modified by the addition of part of the 5′ untranslated region of lamin A (lamin 5′ utr), as well as the coding sequences of a myc tag, a GFP variant (S65T and V163A), and a flexible linker (GGGLDPRVR). The library was initially generated as 20 master pools of 25,000 clones each. (b) Screening the expression library. Starting pools were generated by subdividing each master pool into 20 pools of 2,000 clones. These pools were screened for localized proteins by transfecting each pool into ∼2 × 104 cells grown on a coverslip. The transfection efficiency varied from 20–50% meaning that each clone in the pool was expressed in several cells. Transfected pools were screened visually by epifluorescence microscopy. Pools that gave rise to interesting patterns were retrieved from glycerol stocks and further subdivided and screened until single clones were isolated.
Mentions: To identify proteins localized to different subcellular compartments, a library was constructed with poly(A)-primed cDNA prepared from a human osteosarcoma cell line. The library vector allowed expression of cDNAs in tissue culture cells as COOH-terminal polypeptide fusions to GFP (Fig. 1 a). While GFP is larger than most antibody epitope tags, it forms a tight structure and has been fused to many proteins without disrupting them. Its advantage for a large-scale screen is that it can be directly visualized in transfected cells without the manipulations required for epitope tags.

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