Regulated targeting of the monotopic hairpin membrane protein Erg1 requires the GET pathway

The guided entry of tail-anchored proteins (GET) pathway targets C-terminally anchored transmembrane proteins and protects cells from lipotoxicity. Here, we reveal perturbed ergosterol production in Delta get3 cells and demonstrate the sensitivity of GET pathway mutants to the stero1 synthesis inhibiting drug terbinafine. Our data uncover a key enzyme of sterol synthesis, the hairpin membrane protein squalene monooxygenase (Erg1), as a non-canonical GET pathway client, thus rationalizing the lipotoxicity phenotypes of GET pathway mutants. Get3 recognizes the hairpin targeting element of Erg1 via its classical client-binding pocket. Intriguingly, we find that the GET pathway is especially important for the acute upregulation of Erg1 induced by low sterol conditions. We further identify several other proteins anchored to the endoplasmic reticulum (ER) membrane exclusively via a hairpin as putative clients of the GET pathway. Our findings emphasize the necessity of dedicated targeting pathways for high-efficiency targeting of particular clients during dynamic cellular adaptation and highlight hairpin proteins as a potential novel class of GET clients.