Limits...
Subdomain-specific localization of CLIMP-63 (p63) in the endoplasmic reticulum is mediated by its luminal alpha-helical segment.

Klopfenstein DR, Klumperman J, Lustig A, Kammerer RA, Oorschot V, Hauri HP - J. Cell Biol. (2001)

Bottom Line: The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils.The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization.Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain-specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed alpha-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.

Show MeSH
The luminal segment of CLIMP-63 is required for nuclear envelope exclusion and mobility. COS cells were transiently transfected with wt-GFP (A–C) or Δlumen-GFP (D–F). 48 h after transfection, the cells were fixed with 3% paraformaldehyde, stained with monoclonal antibodies against the ER marker BAP31 (B and E) and analyzed by CLSM. Note that the construct lacking the luminal segment is no longer excluded from the nuclear envelope (D and F). (G) Quantification of FRAP experiments comparing the diffusion and mobility wt-GFP (•) with Δlumen-GFP (○). A representative, transiently transfected cell was photobleached over a 2-μm wide strip covering the ER. Mean fluorescent intensities in the strip were plotted versus time. The prebleach intensity was normalized arbitrarily to 100%. The least square fits to  are indicated as lines. Note the excellent correlation between experimental data and fitting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192027&req=5

Figure 2: The luminal segment of CLIMP-63 is required for nuclear envelope exclusion and mobility. COS cells were transiently transfected with wt-GFP (A–C) or Δlumen-GFP (D–F). 48 h after transfection, the cells were fixed with 3% paraformaldehyde, stained with monoclonal antibodies against the ER marker BAP31 (B and E) and analyzed by CLSM. Note that the construct lacking the luminal segment is no longer excluded from the nuclear envelope (D and F). (G) Quantification of FRAP experiments comparing the diffusion and mobility wt-GFP (•) with Δlumen-GFP (○). A representative, transiently transfected cell was photobleached over a 2-μm wide strip covering the ER. Mean fluorescent intensities in the strip were plotted versus time. The prebleach intensity was normalized arbitrarily to 100%. The least square fits to are indicated as lines. Note the excellent correlation between experimental data and fitting.

Mentions: To explore the function of the luminal segment of CLIMP-63, a mutant lacking this part was generated and compared with the wild-type protein. For reasons of detection, a GFP tag was fused to the COOH terminus of the truncated and wild-type CLIMP-63, resulting in the constructs Δlumen-GFP and wt-GFP, respectively (Fig. 1 A). Because CLIMP-63 is a type II membrane protein, the GFP tag of these constructs is exposed on the luminal side of the ER. Immunofluorescence microscopy of COS cells transfected with wt-GFP showed ER-like staining. No fluorescent ring was seen around the nucleus, indicating that wt-GFP was excluded from the nuclear envelope (Fig. 2, A–C) in the same way as endogenous CLIMP-63 (Klopfenstein et al. 1998). In marked contrast, the Δlumen-GFP showed classical ER staining, including ring-like fluorescence around the nucleus (Fig. 2, D–F), as confirmed by costaining with an antibody against the ER membrane protein BAP31 (Klumperman et al. 1998; Fig. 2B and Fig. E). To establish these staining patterns at the ultrastructural level, immuno-EM was performed using anti-GFP antibodies (Fig. 3) and the data were quantified (Table ). This analysis unequivocally showed that wt-GFP was excluded from the nuclear envelope, whereas high labeling densities were obtained in ER cisternae (Fig. 3 A). Quantification showed that the labeling of the nuclear envelope was not different from background labeling. The labeling ratio of nuclear envelope to ER cisternae was 1:34. Δlumen-GFP, on the other hand, was associated with both ER domains (Fig. 3 B and Table ). The labeling ratio of the nuclear envelope to ER cisternae was 1:4. These morphological results indicate that the luminal segment of CLIMP-63 is required for its subdomain-specific localization in the peripheral ER.


Subdomain-specific localization of CLIMP-63 (p63) in the endoplasmic reticulum is mediated by its luminal alpha-helical segment.

Klopfenstein DR, Klumperman J, Lustig A, Kammerer RA, Oorschot V, Hauri HP - J. Cell Biol. (2001)

The luminal segment of CLIMP-63 is required for nuclear envelope exclusion and mobility. COS cells were transiently transfected with wt-GFP (A–C) or Δlumen-GFP (D–F). 48 h after transfection, the cells were fixed with 3% paraformaldehyde, stained with monoclonal antibodies against the ER marker BAP31 (B and E) and analyzed by CLSM. Note that the construct lacking the luminal segment is no longer excluded from the nuclear envelope (D and F). (G) Quantification of FRAP experiments comparing the diffusion and mobility wt-GFP (•) with Δlumen-GFP (○). A representative, transiently transfected cell was photobleached over a 2-μm wide strip covering the ER. Mean fluorescent intensities in the strip were plotted versus time. The prebleach intensity was normalized arbitrarily to 100%. The least square fits to  are indicated as lines. Note the excellent correlation between experimental data and fitting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192027&req=5

Figure 2: The luminal segment of CLIMP-63 is required for nuclear envelope exclusion and mobility. COS cells were transiently transfected with wt-GFP (A–C) or Δlumen-GFP (D–F). 48 h after transfection, the cells were fixed with 3% paraformaldehyde, stained with monoclonal antibodies against the ER marker BAP31 (B and E) and analyzed by CLSM. Note that the construct lacking the luminal segment is no longer excluded from the nuclear envelope (D and F). (G) Quantification of FRAP experiments comparing the diffusion and mobility wt-GFP (•) with Δlumen-GFP (○). A representative, transiently transfected cell was photobleached over a 2-μm wide strip covering the ER. Mean fluorescent intensities in the strip were plotted versus time. The prebleach intensity was normalized arbitrarily to 100%. The least square fits to are indicated as lines. Note the excellent correlation between experimental data and fitting.
Mentions: To explore the function of the luminal segment of CLIMP-63, a mutant lacking this part was generated and compared with the wild-type protein. For reasons of detection, a GFP tag was fused to the COOH terminus of the truncated and wild-type CLIMP-63, resulting in the constructs Δlumen-GFP and wt-GFP, respectively (Fig. 1 A). Because CLIMP-63 is a type II membrane protein, the GFP tag of these constructs is exposed on the luminal side of the ER. Immunofluorescence microscopy of COS cells transfected with wt-GFP showed ER-like staining. No fluorescent ring was seen around the nucleus, indicating that wt-GFP was excluded from the nuclear envelope (Fig. 2, A–C) in the same way as endogenous CLIMP-63 (Klopfenstein et al. 1998). In marked contrast, the Δlumen-GFP showed classical ER staining, including ring-like fluorescence around the nucleus (Fig. 2, D–F), as confirmed by costaining with an antibody against the ER membrane protein BAP31 (Klumperman et al. 1998; Fig. 2B and Fig. E). To establish these staining patterns at the ultrastructural level, immuno-EM was performed using anti-GFP antibodies (Fig. 3) and the data were quantified (Table ). This analysis unequivocally showed that wt-GFP was excluded from the nuclear envelope, whereas high labeling densities were obtained in ER cisternae (Fig. 3 A). Quantification showed that the labeling of the nuclear envelope was not different from background labeling. The labeling ratio of nuclear envelope to ER cisternae was 1:34. Δlumen-GFP, on the other hand, was associated with both ER domains (Fig. 3 B and Table ). The labeling ratio of the nuclear envelope to ER cisternae was 1:4. These morphological results indicate that the luminal segment of CLIMP-63 is required for its subdomain-specific localization in the peripheral ER.

Bottom Line: The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils.The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization.Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain-specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed alpha-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.

Show MeSH