A novel fractionation method of the rough ER integral membrane proteins; resident proteins versus exported proteins?
We treated the high salt-washed canine pancreatic rough ER (KRM) with 0.18% Triton X-100, separated the extract from the residual membrane (0.18%Tx KRM), and processed the extract with SM-2 beads to recover membrane proteins in proteoliposomes. To focus on integral membrane proteins, KRM, 0.18%Tx KRM and proteoliposomes were subjected to sodium carbonate treatment, and analyzed by 2-D gel electrophoresis. Consequently we found that a distinct group of integral membrane protein of KRM preferentially extracted from the membrane and recovered in proteoliposomes did exist, while majority of KRM integral membrane proteins were fractionated in 0.18%Tx KRM, which retained the basic structure and functions of KRM. Protein identification showed that the former group was enriched with proteins exported from the ER and the latter group comprised mostly of ER resident proteins. This result will potentially affect the prevailing view of the ER membrane structure as well as protein sorting from the ER.
Sakai
Kumiko
K
Department of Cell Biology, Faculty of Medicine, Oita University, Yufu-Shi, Oita, Japan.
Hamanaka
Ryoji
R
Yuki
Hideo
H
Watanabe
Makoto
M
D000818:Animals; D002254:Carbonates / Q000737:chemistry; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D018870:Endoplasmic Reticulum, Rough / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry*; D008565:Membrane Proteins / Q000302:isolation & purification*; D010179:Pancreas / Q000166:cytology; D021381:Protein Transport; D011510:Proteolipids / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D014461:Ultracentrifugation / Q000379:methods
D002254:Carbonates; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; C005686:sodium carbonate