Partitioning to ordered membrane domains regulates the kinetics of secretory traffic

Abstract

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, Retention Using Selective Hooks) 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 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.

Sorting of secretory proteins at the trans-Golgi network by human TGN46

“…Altogether, these studies have led to the suggestion of the hydrophobic matching model, which proposes that gradients in bilayer thickness along the membranes of the secretory pathway contribute/drive the sorting and retention of TM proteins across the secretory pathway. Although there is compelling evidence for a TMD-based mechanism for targeting certain TM proteins to specific cell surface domains (Milovanovic et al, 2015), to localize type-II resident proteins of the cell surface and the Golgi membranes (Munro, 1995; Quiroga et al, 2013), and also to sort the type-III TM protein linker for activation of T-cells (LAT) by raft-mediated partitioning (Diaz-Rohrer et al, 2014; Lorent et al, 2017; Castello-Serrano et al, 2023), it remained unclear how this mechanism contributed to the retention, sorting and lateral segregation of Golgi resident enzymes and their substrates. Interestingly, by using a coarse-grained mathematical model of intra-Golgi transport, Dmitrieff et al showed that experimental data on the Golgi dynamics of secretory cargoes and Golgi residents cannot be explained if the hydrophobic matching mechanism is the sole retention mechanism for Golgi residents (Dmitrieff et al, 2013).…”