Partitioning to ordered membrane domains regulates the kinetics of secretory traffic

• Published in: eLife Vol. 12
• Main Authors: Castello-Serrano, Ivan, Heberle, Frederick A, Diaz-Rohrer, Barbara, Ippolito, Rossana, Shurer, Carolyn R, Lujan, Pablo, Campelo, Felix, Levental, Kandice R, Levental, Ilya
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 05.06.2024; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Adapter proteins; Affinity; Cell Biology; Cell Membrane - metabolism; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Experiments; Golgi apparatus; Golgi Apparatus - metabolism; HeLa Cells; Humans; Kinetics; Lipid rafts; Lipids; Localization; Membrane lipids; Membrane Microdomains - metabolism; Membrane proteins; Membrane Proteins - metabolism; membrane structure; Membrane trafficking; membrane transport; Membranes; Organelles; Physics of Living Systems; Preferences; Probes; Protein folding; Protein Transport; Secretory Pathway; secretory trafficking; Transmembrane domains; secretory trafficking; membrane transport; cell biology; none; physics of living systems; membrane structure
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/elife.89306

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, etention sing elective ooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.