Identification of residues critical for topology inversion of the transmembrane protein TM4SF20 through regulated alternative translocation

Adopting a proper topology is crucial for transmembrane proteins to perform their functions. We previously reported that ceramide regulates a transmembrane protein called TM4SF20 (transmembrane 4 L six family member 20) through topological inversion by altering the direction through which the protein is translocated across membranes during translation. This regulatory mechanism, denoted regulated alternative translocation (RAT), depends on a GXXXN motif present in the first transmembrane helix of TM4SF20. Here, using site-directed mutagenesis, we show that Asn-26 in the motif is crucial for RAT of TM4SF20, as it cannot be replaced even by Gln. In contrast, Gly-22 in the motif could be substituted by other small residues such as Ala and Ser without affecting RAT of TM4SF20. We further demonstrate that the GXXXN motif alone is insufficient to induce RAT of a transmembrane protein because TM4SF4, a relative of TM4SF20 that also contains the motif in the first transmembrane helix, did not undergo RAT. Using TM4SF40 –TM4SF20 chimeras, we identified Pro-29 of TM4SF20 as another important element required for RAT of the protein. Substituting Pro-29 alone did not affect RAT of TM4SF20, whereas replacing Pro-29 together with either Leu-25 or Val-17 of TM4SF20 with the corresponding residues of TM4SF4 abolished RAT of TM4SF20. Because Val-17, Gly-22, Leu-25, Asn-26, and Pro-29 are predicted to reside along the same surface of the transmembrane helix, our results suggest that interactions with other proteins mediated by this surface during translocation may be critical for RAT of TM4SF20.