Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Monday Speaker Abstracts

RAFTING IN A RUSH: MEMBRANE MICRODOMAINS IN SECRETORY TRAFFICKING IVAN CASTELLO SERRANO 1 ; Ilya Levental 1 ; Fred Heberle; Kandice Levental 1 ; Rossana Ippolito 1 ; 1 University of Virginia, Molecular Physiology and Biological Physics, Charlottesville, VA, USA Although significant advances have identified a variety of specific motifs responsible for sub- cellular distribution, such motifs are only present on a small subset of membrane proteins. A potential parallel mechanism for organizing membrane protein traffic is sorting small, dynamic membrane domains of preferentially interacting lipids and proteins, known as lipid rafts, that have been widely implicated in some cellular processes. Our lab has recently defined the structural determinants of preferential protein partitioning into these ordered membrane domains and how this affinity is correlated to a plasma membrane distribution. These observations suggested that sorting and trafficking of membrane proteins can be directed by their affinity for a particular membrane environment. To directly assess the role of membrane microdomains in the secretory pathway, we have taken advantage of a robust tool for synchronized protein traffic, known as RUSH (Retention Using Selective Hooks). Here, tagged proteins are retained in specific organelles by a resident “hook”, where they can be quickly released upon introduction of biotin, allowing direct and quantitative analysis of trafficking rates and destinations by fluorescence microscopy. We applied this system to a library of transmembrane domain (TMD) constructs to evaluate the role of raft affinity in secretory traffic. We find that while TMD- encoded raft affinity is fully sufficient for PM sorting, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which requires specific cytosolic sorting motifs. However, we find that Golgi exit rates are highly raft-dependent, with raft preferring proteins exiting ~2.5-fold faster than mutants with perturbed raft affinity. We rationalize these observations with a mechanistic, predictive model of trafficking through the secretory pathway. These observations highlight a central role for lipid rafts in sorting in the secretory pathway. The proposed model helps to understand how TMD proteins migrate from ER to their final post-Golgi destination.

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