Deletions or Specific Substitutions of a Few Residues in the NH2-terminal Region (Ala3 to Thr9) of Sarcoplasmic Reticulum Ca2+-ATPase Cause Inactivation and Rapid Degradation of the Enzyme Expressed in COS-1 Cells

Takashi Daiho,Kazuo Yamasaki,Hiroshi Suzuki,Tomoyuki Saino,Tohru Kanazawa

doi:10.1074/jbc.274.34.23910

Takashi Daiho, Kazuo Yamasaki + Show 3 more

Open Access

https://doi.org/10.1074/jbc.274.34.23910

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Abstract

Amino acid residues in the NH(2)-terminal region (Glu(2) - Ala(14)) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala(3)-Ser(6) region or deletion of two or more consecutive residues in the Ala(3)-Thr(9) region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala(3)-Ser(6) region caused only a small decrease in the specific Ca(2+) transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca(2+) transport rates. In vitro expression experiments indicated that translation, transcription, and integration into the microsomal membranes were not impaired in the mutants that showed very low expression levels in COS-1 cells. Pulse-chase experiments using [(35)S]methionine/cysteine labeling of transfected COS-1 cells demonstrated that degradation of the mutants showing low expression levels was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of Ala(3). These results suggest that the NH(2)-terminal region (Ala(3) -Thr(9)) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of the SERCA1a protein or stabilization of the correctly folded SERCA1a protein or both.

Highlights

SERCA1a is composed of 10 transmembrane ␣-helices (M1 to M10) and two main cytoplasmic domains, a small loop (Ala132 to Asp237 between M2 and M3) and a large loop (Asn330 to Asn739 between M4 and M5) [1]
The functional role of the NH2-terminal domain is less clear, our recent chemical modification study [15] has suggested that His5 in this domain is located very close to the catalytic site. It was previously shown [16] that deletion of most of the residues (Glu2–His32) in the NH2-terminal domain results in greatly reduced expression in COS-1 cells and inactivation of the enzyme. This raises the possibility that the NH2terminal domain has a region sensitive to the endoplasmic reticulum (ER)-mediated quality control, the machinery of which recognizes and rapidly degrades misfolded proteins and denatured proteins to suppress their cellular expression or accumulation [17, 18]
The results show that deletions or specific substitutions of residues in the Ala3– Thr9 region lead to greatly reduced expression of the mutated SERCA1a proteins and rapid degradation of the expressed SERCA1a proteins

Summary

Introduction

SERCA1a is composed of 10 transmembrane ␣-helices (M1 to M10) and two main cytoplasmic domains, a small loop (Ala132 to Asp237 between M2 and M3) and a large loop (Asn330 to Asn739 between M4 and M5) [1]. The Ca2ϩ transport curve in the presence of 0.5 ␮M thapsigargin with the microsomal membranes expressing the wild-type or mutant SERCA1a was not significantly different from that with control microsomal membranes, which were prepared from COS-1 cells transfected with the pMT2 vector containing no SERCA1a cDNA.

Results

Conclusion