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

Abstract

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.

Keywords: EMC; GET pathway; SND pathway; co-chaperones; protein targeting; tail-anchored proteins.

FIGURE 1

Compiling a list of TA proteins from the human and yeast genomes. (A) A schematic of the pipeline used to gather TA proteins by filtering the Human and Yeast proteomes for TA proteins. (B) A comparison of the TA proteins collected for the analyses here vs previous datasets. (C) Localizations gathered from Uniprot entry Subcellular Localizations (CC) and Gene Ontology Cellular Compartment (GO) annotations. Those with conflicts were resolved by manually parsing the literature to build the final set

FIGURE 2

Investigating properties encoded in the C-terminal residues of TA proteins. For A-F, Jitter plots of property distribution for predicted TA proteins identified as ER (green) or mitochondria (purple) with the best predictive threshold indicated by a dashed red line. Properties visualized are for the C-terminal number of (A) positive residues, (B) negative residues, and (C) net charge and then for (D) TMD length, (E) TMD hydrophobicity, and (F) maximum hydrophobicity of an 18-residue stretch. (G) The AUROC across various hydrophobicity scales for the mean, total, and 18-residue windows of the predicted TMDs

FIGURE 3

Analyzing different geometries of hydrophobic residues in TMDs to improve classification. (A) Alpha-helices and helical wheel plots illustrating the residues selected (orange) for each metric tested, patch, wheel face and segment, showing residues selected and not selected (blue) in each analysis. (B) AUROC values for the metrics illustrated in (A) and total hydrophobicity. (C) Jitter plots as in Figure 2 for the top four hydrophobic metrics: Patch 15 (Kyte & Doolittle scale), Patch 15 (TM Tendency scale), Wheel Face 5 (TM Tendency scale) and Patch 11 (Kyte & Doolittle scale). Red dashed line indicates the best predictive threshold. (D) 2D comparison plot of total hydrophobicity (y-axis) and a Wheel Face 5 (TM Tendency scale) (x-axis). TA proteins are colored by localization, ER ( green), mitochondria (purple), Unknown ( gray), both mitochondria and ER (blue), and peroxisome (orange). TA proteins selected for experimental determination of localizations are marked squares. Dashed lines indicate best predictive threshold

FIGURE 4

Localization of unknown yeast TA proteins. The ER (magenta panel) and mitochondria (cyan panel) were labeled with tdTomato and BFP, respectively. TA protein localization was visualized by GFP (yellow panel) and colocalization was determined by overlap (merge panel). The ratio of the number of cells with the TA protein localizing to the ER vs the mitochondria are noted in the merge image. Numbered as in Figure 3D with labels colored based on their determined localizations: ER (green) and mitochondria (purple). TA proteins include (A) two mitochondrial TA proteins with known localizations and (B) 15 with unknown localizations

FIGURE 5

A hydrophobic Wheel Face metric of 5 or 7 residues best separates ER and mitochondria TA proteins. (A) A ranking of the five best performing hydrophobicity metrics compared to the TMD hydrophobicity metrics of the appropriate hydrophobicity scales (TM Tendency, Fauchere & Pliska and Kyte and Doolittle). The number of correctly predicted localizations as well as the final AUROC scores are used to assess the effectiveness of each metric. The total number of correctly classified yeast TA proteins is also noted. The two metrics directly compared in the 2D comparison plot in (B) are highlighted in blue (TM Tendency, Wheel Face 5, x-axis) and red (TM Tendency, TMD, y-axis). Hydrophobicities are plotted and TA proteins are colored as they were in Figure 3D. Newly determined localizations from Figure 4 (black outlined) and Weill et al (squares) are filled in with the appropriate colors, ER (green), mitochondria (purple) and peroxisome (orange)

FIGURE 6

Human ER and mitochondrial TA proteins can be separated by the most hydrophobic 11 residues segment. (A) A table of the with the AUROC values of the best performing hydrophobicity metrics and the overall TMD hydrophobicity, along with their ranking. The number of total misclassified proteins are separated by ER and mitochondria TA proteins. (B) 2D comparison for the human dataset of TMD hydrophobicity and Patch 11 metrics using the Kyte and Doolittle scale. Hydrophobicities are plotted and TA proteins are colored as in Figure 3D. Unknown TA proteins are not plotted

FIGURE 7

Combining a hydrophobicity and C-terminal charge metric results in a more effective predictor. The most hydrophobic 11 amino acid segment of all human TA protein TMDs with known localizations to either the ER (green) or mitochondria (purple) was calculated using the Kyte and Doolittle scale and plotted along the x-axis. The number of positive charge residues was counted and plotted along the y-axis. The best fit cut-off for the hydrophobicity metric (blue dotted line) and charge metric (red dotted line) are marked. The number of ER and mitochondria TA proteins captured in each step is denoted in the corresponding quadrant