S-palmitoylation determines TMEM55B-dependent positioning of lysosomes.

The spatiotemporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo. TMEM55B (also known as PIP4P1), as an integral lysosomal membrane protein, is a component of such a complex that mediates the retrograde transport of lysosomes by establishing interactions with the cytosolic scaffold protein JIP4 (also known as SPAG9) and dynein-dynactin. Here, we show that TMEM55B and its paralog TMEM55A (PIP4P2) are S-palmitoylated proteins that are lipidated at multiple cysteine residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes retention of the mutated protein in the Golgi. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor protein-dependent sorting of TMEM55B. Our data suggest a critical role for S-palmitoylation in the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

R17C Mutation in Photoreceptor Disc-Specific Protein, PRCD, Results in Additional Lipidation Altering Protein Stability and Subcellular Localization.

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases.

Differential S-palmitoylation of the human and rodent ß3-adrenergic receptors.

With few reported exceptions, G protein-coupled receptors (GPCRs) are modified by Cys palmitoylation (S-palmitoylation). In multiple GPCRs, S-palmitoylation targets a canonical site within the C-terminal cytoplasmic tail adjacent to the C terminus of the seventh transmembrane domain, but modification of additional sites is exemplified by the ß-adrenergic receptors (ßARs). The ß1AR is S-palmitoylated at a second, more distal site within the C-terminal tail, and the ß2AR is modified at a second site within the third intracellular loop, neither of which is conserved in other ßAR isoforms. The functional roles of S-palmitoylation of disparate sites are incompletely characterized for any GPCR family. Here, we describe S-palmitoylation of the ß3AR. We compared mouse and human ß3ARs and found that both were S-palmitoylated at the canonical site within the C-terminal tail, Cys-358 and Cys-361/363 in mouse and human ß3ARs, respectively. Surprisingly, the human ß3AR was S-palmitoylated at two additional sites, Cys-153 and Cys-292 within the second and third intracellular loops, respectively. Cys-153 is apparently unique to the human ß3AR, and Cys-292 is conserved primarily in primates. Mutational substitution of C-tail Cys in human but not mouse ß3ARs resulted in diminished ligand-induced cAMP production. Substitution of Cys-153, Cys-292, or Cys-361/363 within the human ß3AR diminished membrane-receptor abundance, but only Cys-361/363 substitution diminished membrane-receptor half-life. Thus, S-palmitoylation of different sites differentially regulates the human ß3AR, and differential S-palmitoylation distinguishes human and rodent ß3ARs, potentially contributing to species-specific differences in the clinical efficacy of ß3AR-directed pharmacological approaches to disease.

A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS.

Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein-protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3H- or 14C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin-assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective. Graphical abstract.

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells.

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein-protein or protein-membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.

S-palmitoylation (S-acylation) is emerging as an important dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labeling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labeled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide (NEM), to prevent non-specific detection. This in turn necessitates multiple precipitation-based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal, or protein aggregation. These combine to reduce the sensitivity, reliability, and accuracy of these assays, which also require a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of NEM by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction, it is possible to greatly improve sensitivity and accuracy while reducing the hands-on time and overall time required for the assay.

SwissPalm: Protein Palmitoylation database.

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.