Sequence‐based features that are determinant for tail‐anchored membrane protein sorting in eukaryotes

The correct targeting and insertion of tail‐anchored (TA) integral membrane proteins is critical for cellular homeostasis. TA proteins are defined by a hydrophobic transmembrane domain (TMD) at their C‐terminus and are targeted to either the ER or mitochondria. Derived from experimental measurements of a few TA proteins, there has been little examination of the TMD features that determine localization. As a result, the localization of many TA proteins are misclassified by the simple heuristic of overall hydrophobicity. Because ER‐directed TMDs favor arrangement of hydrophobic residues to one side, we sought to explore the role of geometric hydrophobic properties. By curating TA proteins with experimentally determined localizations and assessing hypotheses for recognition, we bioinformatically and experimentally verify that a hydrophobic face is the most accurate singular metric for separating ER and mitochondria‐destined yeast TA proteins. A metric focusing on an 11 residue segment of the TMD performs well when classifying human TA proteins. The most inclusive predictor uses both hydrophobicity and C‐terminal charge in tandem. This work provides context for previous observations and opens the door for more detailed mechanistic experiments to determine the molecular factors driving this recognition. Despite the knowledge of pathways identified for targeting tail‐anchored (TA) membrane proteins to the ER, as opposed to the mitochondria, the properties of the TA sequence that are recognized for sorting remain to be understood with many localizations currently ambiguous at the sequence level. Utilizing bioinformatics and cellular imaging, we demonstrate that TA proteins directed to the ER membrane contain specific hydrophobic features that are determining factors, along with C‐terminal charge properties, for localization.