Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers.
The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca(2+)-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.
Kaakinen
Mika
M
Department of Anatomy and Cell Biology, P.O. Box 5000 (Aapistie 7), FIN-90014 University of Oulu, Finland.
Papponen
Hinni
H
Metsikkö
Kalervo
K
D000818:Animals; D015415:Biomarkers; D002592:Cetomacrogol / Q000378:metabolism; D018076:DNA, Complementary; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases / Q000378:metabolism; D021962:Membrane Microdomains / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D012995:Solubility
D015415:Biomarkers; D018076:DNA, Complementary; D009124:Muscle Proteins; D049452:Green Fluorescent Proteins; D002592:Cetomacrogol; D006602:Hexosyltransferases; C526972:Dad1 protein, rat; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases