Regulation of Immature Protein Dynamics in the Endoplasmic Reticulum

Kamada, Asako; Nagaya, Hisao; Tamura, Taku; Kinjo, Masataka; Jin, Hongfang; Yamashita, Toshiharu; Jimbow, Kowichi; Kanoh, Hideo; Wada, Ikuo

doi:10.1074/jbc.m401403200

Cited by 20 publications

(34 citation statements)

References 57 publications

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“…For this purpose, we used BODIPY FL-pepstatin A (FLpepstatin) in order to avoid artifacts due to immunostaining after permeabilization. Previously, we showed that active tyrosinase is delivered to structures highly enriched in the fluorescent pepstatin (24). Lysosomes were marked by LysoTracker Red DND-99, a lysosome-specific FIGURE 6.…”

Section: Reduced Expression Of D12 Induces Formation Of Lipofuscin Anmentioning

confidence: 99%

“…These plasmids were introduced into cells using either FuGENE6 (Roche Applied Science) or bead loading (fluorescence correlation spectroscopy analysis) (23). For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin for 1 min on ice or fixed and permeabilized with methanol at Ϫ20°C essentially as described (24). After staining, the specimens were observed with confocal microscopy using an LSM PASCAL or LSM510metaConfoCor2 (Carl Zeiss).…”

Section: Modified Fl-rex To Search For Proteins In the Secretory Pathmentioning

confidence: 99%

“…The images were taken using cooled CCD Cascade 512B (Photometrics) and processed with MetaMorph (Universal Imaging). The diffusion time of D12-(1-80)-mRFP1 in living cells was estimated by using fluorescence correlation spectroscopy as described previously (24).…”

Section: Modified Fl-rex To Search For Proteins In the Secretory Pathmentioning

confidence: 99%

“…Unbroken cells and nuclei were removed by centrifugation at 1,200 ϫ g for 5 min. The postnuclear supernatant was loaded on preformed Nycodenz (Sigma) gradients, which were prepared for the HITACHI S52ST rotor from initial discontinuous gradients (24,19,15, and 10% Nycodenz in 10 mM Tris-HCl (pH 7.5), 3 mM KCl, and 1 mM EDTA) that were allowed to diffuse in a horizontal position for 45 min at room temperature and then centrifuged for 4 h at 37,000 rpm in a Himac CS100GXL (Hitachi) ultracentrifuge to generate a nonlinear density gradient profile. The postnuclear supernatant was loaded on top of the gradient and centrifuged for 4 h at 37,000 rpm.…”

Section: Modified Fl-rex To Search For Proteins In the Secretory Pathmentioning

confidence: 99%

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Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

Okumura

Hatsuzawa

Tamura

et al. 2006

Journal of Biological Chemistry

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The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to ␣-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomallysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

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Section: Reduced Expression Of D12 Induces Formation Of Lipofuscin Anmentioning

confidence: 99%

Section: Modified Fl-rex To Search For Proteins In the Secretory Pathmentioning

confidence: 99%

Section: Modified Fl-rex To Search For Proteins In the Secretory Pathmentioning

confidence: 99%

Section: Modified Fl-rex To Search For Proteins In the Secretory Pathmentioning

confidence: 99%

See 2 more Smart Citations

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

Okumura

Hatsuzawa

Tamura

et al. 2006

Journal of Biological Chemistry

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“…Introduction of GFPfused eIF5 expression plasmids by using siliconized glass microbeads into cells was described previously (21). For fluorescence correlation spectroscopy (FCS) analysis, a ConfoCor2 instrument (Zeiss) was used and analysis carried out as described (22).…”

Section: Analysis Of Protein Complexes By Sucrose Density Centrifugatmentioning

confidence: 99%

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Homma

Wada

Suzuki

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser͞Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G 1 and G2͞M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5͞eIF2͞eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation. C asein kinase 2 (CK2) (1-4) is composed of two subunits, ␣ or ␣Ј and ␤, which combine to form a native ␣ 2 ␤ 2 tetramer. Disruption of the catalytic subunits (␣ and ␣Ј) is lethal in Saccharomyces cerevisiae (5) and disruption of the regulatory ␤ subunit in mice leads to early embryonic lethality (6). CK2 phosphorylates a range of cellular targets in a variety of subcellular sites and appears to be highly pleiotropic; it is involved in many key biological functions, including growth and cell cycle control (7), signal transduction (3), circadian rhythms (8, 9), and gene expression (10, 11). CK2 is also a stress-activated kinase and might participate in the transduction of survival signals to avoid damage by mutagenic UV radiation (12, 13). An important role for CK2 in promoting cell proliferation and transformation has been indicated by several studies. In mammalian systems, its targeted overexpression in mice results in the development of T cell lymphoma and mammary tumorigenesis (5). Despite these findings, there is still much uncertainty regarding the activation of CK2 in response to stimuli (14). The mechanism by which it is regulated and its precise function in cell cycle progression and proliferation is still poorly understood.CK2 activity and stability are believed to be regulated in part by holoenzyme formation via a self-assembly mechanism and by phosphorylation. Phosphorylation by p34 cdc2 of the catalytic ␣ subunit at the C-terminal domain occurs in ...

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Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy

2007

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The diffusion properties of EGFP-hGRa and mutants C421G, A458T and I566 in living cells were analyzed. The wild type and mutants C421G and A458T translocated from the cytoplasm to the nucleus after addition of Dex; however, the Brownian motions of the proteins were different. The diffusion constant of wild-type GRa after addition of Dex slowed to 15.6% of that in the absence of Dex, whereas those of A458T and C421G slowed to 34.8% and 61.7%, respectively. This is the first report that dimer formation is less important than the binding activity of GRa to GRE in the living cell.

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Regulation of Immature Protein Dynamics in the Endoplasmic Reticulum

Cited by 20 publications

References 57 publications

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy

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