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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.

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Overexpression of CLIMP-63 results in formation of ER clusters in restricted areas of the cytoplasm. COS cells were transiently transfected with wt-GFP and subjected to ultrathin cryosectioning followed by immunolabeling with anti-GFP and protein A–gold. (A) The cell at the left expresses high levels of wt-GFP as judged by the high density of gold particles and displays clustered ER (arrows). The cell at the right expresses no or undetectable levels of wt-GFP and shows nonclustered ER (arrows). (B) Another example of an ER cluster in a cell with high expression of wt-GFP. N, nucleus; P, plasma membrane. Bars: (A) 1 μm; (B) 500 nm.
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Figure 5: Overexpression of CLIMP-63 results in formation of ER clusters in restricted areas of the cytoplasm. COS cells were transiently transfected with wt-GFP and subjected to ultrathin cryosectioning followed by immunolabeling with anti-GFP and protein A–gold. (A) The cell at the left expresses high levels of wt-GFP as judged by the high density of gold particles and displays clustered ER (arrows). The cell at the right expresses no or undetectable levels of wt-GFP and shows nonclustered ER (arrows). (B) Another example of an ER cluster in a cell with high expression of wt-GFP. N, nucleus; P, plasma membrane. Bars: (A) 1 μm; (B) 500 nm.

Mentions: To test if the entire luminal segment is required for nuclear envelope exclusion and immobilization, we made deletions in the luminal part of CLIMP-63 (Fig. 1 A) and tested the constructs in FRAP experiments. In these constructs the cytosolic segment of CLIMP-63 was replaced by GFP. This replacement was required to exclude a possible interference by the cytosolic microtubule–binding segment. COS cells transfected with the GFP-Δcytoplasmic construct that carries the entire luminal segment of CLIMP-63 (Fig. 1 A) showed reticular ER staining without a nuclear ring (Fig. 4 A) comparable to wt-GFP. In addition to reticular ER staining, ∼25% of the transfected cells (n = 864) exhibited highly fluorescent perinuclear areas (Fig. 4 D). Other ER markers, such as BAP31 (Klumperman et al. 1998), colocalized with these areas (Fig. 4 E, merger in F). Analysis by immuno-EM showed that these areas of intense fluorescence correspond to regions of densely packed ER elements (Fig. 5). The labeling for CLIMP-63 in clustered and nonclustered ER elements was comparable. Obviously, CLIMP-63 can concentrate ER membranes by its luminal segment in the absence of the cytoplasmic segment. The exclusion of the GFP-Δcytoplasmic construct from the nuclear membrane was confirmed by immuno-EM (Fig. 3 C and Table ). Unlike GFP-Δcytoplasmic, all luminal deletion constructs (Fig. 1 A) had access to the nuclear envelop (Fig. 4 and Fig. 7). One of these constructs, Δ218–601, was also studied by immuno-EM, which confirmed its dual localization in both subdomains of the ER (Fig. 3 D and Table ). These results suggest that exclusion of CLIMP-63 from the nuclear envelope depends on an intact luminal segment.


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)

Overexpression of CLIMP-63 results in formation of ER clusters in restricted areas of the cytoplasm. COS cells were transiently transfected with wt-GFP and subjected to ultrathin cryosectioning followed by immunolabeling with anti-GFP and protein A–gold. (A) The cell at the left expresses high levels of wt-GFP as judged by the high density of gold particles and displays clustered ER (arrows). The cell at the right expresses no or undetectable levels of wt-GFP and shows nonclustered ER (arrows). (B) Another example of an ER cluster in a cell with high expression of wt-GFP. N, nucleus; P, plasma membrane. Bars: (A) 1 μm; (B) 500 nm.
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Related In: Results  -  Collection

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Figure 5: Overexpression of CLIMP-63 results in formation of ER clusters in restricted areas of the cytoplasm. COS cells were transiently transfected with wt-GFP and subjected to ultrathin cryosectioning followed by immunolabeling with anti-GFP and protein A–gold. (A) The cell at the left expresses high levels of wt-GFP as judged by the high density of gold particles and displays clustered ER (arrows). The cell at the right expresses no or undetectable levels of wt-GFP and shows nonclustered ER (arrows). (B) Another example of an ER cluster in a cell with high expression of wt-GFP. N, nucleus; P, plasma membrane. Bars: (A) 1 μm; (B) 500 nm.
Mentions: To test if the entire luminal segment is required for nuclear envelope exclusion and immobilization, we made deletions in the luminal part of CLIMP-63 (Fig. 1 A) and tested the constructs in FRAP experiments. In these constructs the cytosolic segment of CLIMP-63 was replaced by GFP. This replacement was required to exclude a possible interference by the cytosolic microtubule–binding segment. COS cells transfected with the GFP-Δcytoplasmic construct that carries the entire luminal segment of CLIMP-63 (Fig. 1 A) showed reticular ER staining without a nuclear ring (Fig. 4 A) comparable to wt-GFP. In addition to reticular ER staining, ∼25% of the transfected cells (n = 864) exhibited highly fluorescent perinuclear areas (Fig. 4 D). Other ER markers, such as BAP31 (Klumperman et al. 1998), colocalized with these areas (Fig. 4 E, merger in F). Analysis by immuno-EM showed that these areas of intense fluorescence correspond to regions of densely packed ER elements (Fig. 5). The labeling for CLIMP-63 in clustered and nonclustered ER elements was comparable. Obviously, CLIMP-63 can concentrate ER membranes by its luminal segment in the absence of the cytoplasmic segment. The exclusion of the GFP-Δcytoplasmic construct from the nuclear membrane was confirmed by immuno-EM (Fig. 3 C and Table ). Unlike GFP-Δcytoplasmic, all luminal deletion constructs (Fig. 1 A) had access to the nuclear envelop (Fig. 4 and Fig. 7). One of these constructs, Δ218–601, was also studied by immuno-EM, which confirmed its dual localization in both subdomains of the ER (Fig. 3 D and Table ). These results suggest that exclusion of CLIMP-63 from the nuclear envelope depends on an intact luminal segment.

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.

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