Palmitoylation regulates raft affinity for the majority of integral raft proteins

Abstract

The physical basis for protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by nonionic detergents. We have used giant plasma membrane vesicles, a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to measure the partitioning of the transmembrane linker for activation of Tcells (LAT). LATenrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines and could be enhanced by oligomerization. This palmitoylation requirement was also shown to regulate raft phase association for the majority of integral raft proteins. Because cysteine palmitoylation is the only lipid modification that has been shown to be reversibly regulated, our data suggest a role for palmitoylation as a dynamic raft targeting mechanism for transmembrane proteins.phase separation | raft partitioning | posttranslational modification | GPI-anchored protein P osttranslational modifications allow for rapid modulation of protein structure, localization, and function. An important class is lipid modifications, which includes the addition of GPI anchors, sterols, as well as single saturated and/or unsaturated fatty acids. The cellular purpose of protein lipidation is often to anchor the modified polypeptide to membranes; however, the functional significance of the widespread S acylation of transmembrane (TM) proteins remains unclear. Additionally, S acylation, often referred to as "palmitoylation" due to the addition of a cysteine-linked palmitate, is the only protein lipidation under dynamic enzymatic regulation, implying a potentially important regulatory role (1).Adapting a recently developed experimental system for measuring protein partitioning between coexisting fluid domains in cell-derived isolated plasma membranes, we find that palmitoylation regulates raft phase affinity of LAT (linker for activation of T cells), a critical adaptor in immune system signaling. This finding extends to the majority of raft phase partitioning proteins, suggesting a general role for protein palmitoylation in dynamic regulation of raft association. Although previous studies (2-7) have implicated palmitoylation in regulation of detergent resistance of TM proteins, the indirect and controversial nature of these experiments has limited their applicability in assigning raft affinity. The results presented here directly demonstrate and quantify the vital role of palmitoylation in partitioning of TM proteins to raft phase domains of isolated plasma membranes (PM). ResultsLAT Partitioning Is Disrupted by DTT. Giant plasma membrane vesicles (GPMVs) are cell-and cytoskeleton-detached, organellefree PM blebs that maintain the protein (8) and lipid (9) diversity of the native membrane and separate into two liquid phases (10) with different order (11) mirroring the behavior of pure lipid liposomes (12). Remarkably, phase separation sorts membrane com...

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…Os-Rac1 belongs to the type II Rac/ROP subgroup, whose members are attached to the PM by palmitoylation of a conserved GC-CG box (Lavy et al, 2002;Levental et al, 2010). Palmitoylation plays a key role in targeting proteins to PM microdomains (Levental et al, 2010). Arabidopsis ROP6, a type I ROP possessing a CaaL box motif, is primarily geranylgeranylated and, when activated, accumulates in DRMs with transient palmitoylation (Sorek et al, 2007(Sorek et al, , 2010.…”

“…In this study, we provided evidence that endogenous Os-Rac1 is transiently localized to PM microdomains at an early stage of the chitin response. Os-Rac1 belongs to the type II Rac/ROP subgroup, whose members are attached to the PM by palmitoylation of a conserved GC-CG box (Lavy et al, 2002;Levental et al, 2010). Palmitoylation plays a key role in targeting proteins to PM microdomains (Levental et al, 2010).…”

Recruitment of activating NK‐cell receptors 2B4 and NKG2D to membrane microdomains in mammalian cells is dependent on their transmembrane regions

“…While it is true that data from such assays have to be regarded with care, they still contain meaningful information. The localization of membrane components in a DRM assay is not random: glycosylphosphatidylinositol anchored proteins are enriched in DRMs [38] similar to other proteins with different modifications, for example, palmitoylation [39]. We and others have previously shown that inhibitory NK-cell receptors are not recruited to DRMs after receptor engagement [16,18].…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…The best-studied PTM and probably the most notorious is phosphorylation, however acylation, also known as fatty acylation or lipidation, is emerging as a widespread PTM (Nadolski and Linder, 2007). In particular, palmitoylation might have been underestimated for decades due to its labile nature that can be disrupted by reducing conditions (Levental et al, 2010) and due to the low sensitivity of radioactive palmitate labelling that necessitated weeks to months of exposure of autoradiograms. However, the recent development of new technologies to capture and identify palmitoylated proteins (Hannoush and Sun, 2010) has led to the realisation that this PTM is widely used not only by eukaryotes, but also by viruses and bacteria that are able to subvert the host palmitoylation machinery .…”

“…In contrast, S-palmitoylation is a labile thioester linkage arising from the post-translational addition of a 16-carbon saturated fatty acid (palmitate) to a cysteine residue that can be within either soluble or integral membrane proteins (Linder and Deschenes, 2007). Soluble proteins are profoundly modified by saturated lipids (myristate and palmitate) that promote their insertion into microdomains or lipid rafts (Levental et al, 2010;Yang et al, 2010) (Fig. 1C).…”