Use of carbonate extraction in analyzing moderately hydrophobic transmembrane proteins in the mitochondrial inner membrane

Resistance to sodium carbonate extraction is regarded as a canonical way to distinguish integral membrane proteins (MPs) from other membrane-associated proteins. However, it has been observed that carbonate extraction releases some mitochondrial integral MPs. Here, by analyzing both artificially designed and native mitochondrial inner MPs containing transmembrane domains (TMDs) of different hydrophobicities, we show that carbonate treatment can release moderately hydrophobic TMDs from the mitochondrial inner membrane. These results suggest that resistance and sensitivity to carbonate extraction may be interpreted with caution when analyzing the nature of mitochondrial inner MPs.