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Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation.

Yamaguchi A, Hori O, Stern DM, Hartmann E, Ogawa S, Tohyama M - J. Cell Biol. (1999)

Bottom Line: The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER.Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress.These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita City, Osaka 565-0871, Japan.

ABSTRACT
Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61alpha and Sec61beta, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

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Immunostaining of BHK cells transfected with the FLAG-tagged pcDNA/SERP1/RAMP4 using anti-FLAG antibody (a), anti-PDI antibody (b), and both (c). SERP1/RAMP4-transfected BHK cells were fixed in 0.1% NP-40/4% paraformaldehyde solution and subjected to the immunostaining protocol (see text). Sites of primary antibody binding were visualized with TRITC-conjugated anti–mouse antibody for FLAG epitope (a), FITC conjugated anti–rabbit antibody for PDI (b), or using both detection systems (c). Upper panels show lower magnification and lower panels higher magnification. Note that the two epitopes colocalize throughout the interior, and even in peripheral regions of the cells.
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Figure 3: Immunostaining of BHK cells transfected with the FLAG-tagged pcDNA/SERP1/RAMP4 using anti-FLAG antibody (a), anti-PDI antibody (b), and both (c). SERP1/RAMP4-transfected BHK cells were fixed in 0.1% NP-40/4% paraformaldehyde solution and subjected to the immunostaining protocol (see text). Sites of primary antibody binding were visualized with TRITC-conjugated anti–mouse antibody for FLAG epitope (a), FITC conjugated anti–rabbit antibody for PDI (b), or using both detection systems (c). Upper panels show lower magnification and lower panels higher magnification. Note that the two epitopes colocalize throughout the interior, and even in peripheral regions of the cells.

Mentions: Immunostaining of BHK cells transiently transfected with FLAG-tagged SERP1/RAMP4 displayed an overlapping distribution of SERP1/RAMP4 antigen and the ER marker PDI (Fig. 3, a–c). Although SERP1/RAMP4 has a putative n-glycosylation site at its NH2 terminus, treatment of SERP1/RAMP4-transfected cells with tunicamycin or incubation of SERP1/RAMP4 protein itself with endoglycosidase H revealed no change in SERP1/RAMP4 (with respect to either its subcellular distribution or migration on SDS-PAGE), suggesting that glycosylation had not occurred (data not shown).


Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation.

Yamaguchi A, Hori O, Stern DM, Hartmann E, Ogawa S, Tohyama M - J. Cell Biol. (1999)

Immunostaining of BHK cells transfected with the FLAG-tagged pcDNA/SERP1/RAMP4 using anti-FLAG antibody (a), anti-PDI antibody (b), and both (c). SERP1/RAMP4-transfected BHK cells were fixed in 0.1% NP-40/4% paraformaldehyde solution and subjected to the immunostaining protocol (see text). Sites of primary antibody binding were visualized with TRITC-conjugated anti–mouse antibody for FLAG epitope (a), FITC conjugated anti–rabbit antibody for PDI (b), or using both detection systems (c). Upper panels show lower magnification and lower panels higher magnification. Note that the two epitopes colocalize throughout the interior, and even in peripheral regions of the cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168098&req=5

Figure 3: Immunostaining of BHK cells transfected with the FLAG-tagged pcDNA/SERP1/RAMP4 using anti-FLAG antibody (a), anti-PDI antibody (b), and both (c). SERP1/RAMP4-transfected BHK cells were fixed in 0.1% NP-40/4% paraformaldehyde solution and subjected to the immunostaining protocol (see text). Sites of primary antibody binding were visualized with TRITC-conjugated anti–mouse antibody for FLAG epitope (a), FITC conjugated anti–rabbit antibody for PDI (b), or using both detection systems (c). Upper panels show lower magnification and lower panels higher magnification. Note that the two epitopes colocalize throughout the interior, and even in peripheral regions of the cells.
Mentions: Immunostaining of BHK cells transiently transfected with FLAG-tagged SERP1/RAMP4 displayed an overlapping distribution of SERP1/RAMP4 antigen and the ER marker PDI (Fig. 3, a–c). Although SERP1/RAMP4 has a putative n-glycosylation site at its NH2 terminus, treatment of SERP1/RAMP4-transfected cells with tunicamycin or incubation of SERP1/RAMP4 protein itself with endoglycosidase H revealed no change in SERP1/RAMP4 (with respect to either its subcellular distribution or migration on SDS-PAGE), suggesting that glycosylation had not occurred (data not shown).

Bottom Line: The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER.Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress.These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita City, Osaka 565-0871, Japan.

ABSTRACT
Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61alpha and Sec61beta, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

Show MeSH
Related in: MedlinePlus