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Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore.

Ohba T, Schirmer EC, Nishimoto T, Gerace L - J. Cell Biol. (2004)

Bottom Line: However, increasing the size of either domain by 47 kD strongly inhibited movement.Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect.Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

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Schematic diagram of protein constructs. The standard reporter plasmid encoded residues 401–452 of LAP2β (LAP2β-TM, which comprises a short cytosolic region and its transmembrane/lumenal domains) fused to GFP and FRB as shown. In some cases, PK was fused to the lumenal (Reporter-PK (L)) or the cytoplasmic (Reporter-PK(C)) side of the reporter. The “protein trap” plasmid encoded the nucleoplasmic domain of LAP2β (residues 1–401; LAP2β-NP) and three tandem copies of FKBP. The molecular masses of the various protein segments are indicated (bottom).
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fig1: Schematic diagram of protein constructs. The standard reporter plasmid encoded residues 401–452 of LAP2β (LAP2β-TM, which comprises a short cytosolic region and its transmembrane/lumenal domains) fused to GFP and FRB as shown. In some cases, PK was fused to the lumenal (Reporter-PK (L)) or the cytoplasmic (Reporter-PK(C)) side of the reporter. The “protein trap” plasmid encoded the nucleoplasmic domain of LAP2β (residues 1–401; LAP2β-NP) and three tandem copies of FKBP. The molecular masses of the various protein segments are indicated (bottom).

Mentions: We have developed an assay to analyze the movement of integral membrane proteins from the peripheral ER to the INM in real time. The assay uses the ability of the cell-permeable small molecule rapamycin to mediate high affinity dimerization between FKBP and FRB (Chen et al., 1995; Klemm et al., 1997). One component of this assay is a reporter protein consisting of (from the NH2 to the COOH terminus) the 11-kD FRB (Chen et al., 1995), amino acid residues 401–452 of lamina-associated polypeptide 2β (LAP2β) containing its membrane-insertion domain (LAP2β-TM; Furukawa et al., 1995), and GFP (Fig. 1). Because LAP2β is a type II integral protein of the INM (Furukawa et al., 1995), this construct gives rise to a transmembrane protein with FRB located in the cytosol and GFP situated in the ER lumen. Additional versions of the reporter construct contained a 47-kD domain from chicken muscle pyruvate kinase (PK; Soullam and Worman, 1995) added to the lumenal or cytosolic side of the transmembrane segment. A second component of the assay was a nucleus-localized protein “trap” that lacked membrane-spanning sequences. This consisted of three tandem repeats of the 12-kD FKBP domain (Klemm et al., 1997) fused to amino acids 1–396 of LAP2β (LAP2β-NP), which contains the LAP2β lamin-binding domain and which is strongly localized to the nucleus of transfected cells (Furukawa et al., 1995).


Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore.

Ohba T, Schirmer EC, Nishimoto T, Gerace L - J. Cell Biol. (2004)

Schematic diagram of protein constructs. The standard reporter plasmid encoded residues 401–452 of LAP2β (LAP2β-TM, which comprises a short cytosolic region and its transmembrane/lumenal domains) fused to GFP and FRB as shown. In some cases, PK was fused to the lumenal (Reporter-PK (L)) or the cytoplasmic (Reporter-PK(C)) side of the reporter. The “protein trap” plasmid encoded the nucleoplasmic domain of LAP2β (residues 1–401; LAP2β-NP) and three tandem copies of FKBP. The molecular masses of the various protein segments are indicated (bottom).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172626&req=5

fig1: Schematic diagram of protein constructs. The standard reporter plasmid encoded residues 401–452 of LAP2β (LAP2β-TM, which comprises a short cytosolic region and its transmembrane/lumenal domains) fused to GFP and FRB as shown. In some cases, PK was fused to the lumenal (Reporter-PK (L)) or the cytoplasmic (Reporter-PK(C)) side of the reporter. The “protein trap” plasmid encoded the nucleoplasmic domain of LAP2β (residues 1–401; LAP2β-NP) and three tandem copies of FKBP. The molecular masses of the various protein segments are indicated (bottom).
Mentions: We have developed an assay to analyze the movement of integral membrane proteins from the peripheral ER to the INM in real time. The assay uses the ability of the cell-permeable small molecule rapamycin to mediate high affinity dimerization between FKBP and FRB (Chen et al., 1995; Klemm et al., 1997). One component of this assay is a reporter protein consisting of (from the NH2 to the COOH terminus) the 11-kD FRB (Chen et al., 1995), amino acid residues 401–452 of lamina-associated polypeptide 2β (LAP2β) containing its membrane-insertion domain (LAP2β-TM; Furukawa et al., 1995), and GFP (Fig. 1). Because LAP2β is a type II integral protein of the INM (Furukawa et al., 1995), this construct gives rise to a transmembrane protein with FRB located in the cytosol and GFP situated in the ER lumen. Additional versions of the reporter construct contained a 47-kD domain from chicken muscle pyruvate kinase (PK; Soullam and Worman, 1995) added to the lumenal or cytosolic side of the transmembrane segment. A second component of the assay was a nucleus-localized protein “trap” that lacked membrane-spanning sequences. This consisted of three tandem repeats of the 12-kD FKBP domain (Klemm et al., 1997) fused to amino acids 1–396 of LAP2β (LAP2β-NP), which contains the LAP2β lamin-binding domain and which is strongly localized to the nucleus of transfected cells (Furukawa et al., 1995).

Bottom Line: However, increasing the size of either domain by 47 kD strongly inhibited movement.Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect.Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

Show MeSH