N‐terminal dual lipidation‐coupled molecular targeting into the primary cilium

Abstract

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders.

Transport of Ciliary Membrane Proteins

“…For example, Unc-119 is responsible for transporting myristoylated NPHP3 and Cystin, and ARL3-GTP releases myristoylated cargo from UNC119 (Wright et al, 2011). A Golgi-localized palmitoyltransferase, DHHC-21, is essential to the palmitoylation-dependent transport of N-terminal dual-acylation proteins to cilia (Kumeta et al, 2018).…”

PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion

“…This finding hints towards a crucial role that the minimal structural organization of PCARE has in its ciliary localization and further work is required to establish the exact role of the coiled coil domain in PCARE trafficking. Moreover, myristoylation and palmitoylation were predicted at the N-terminus of PCARE, modifications that are known to play a role in ciliary trafficking in Trypanosoma brucei and in eukaryotic cells ( 19 , 20 ). N-myristoylation on p.Gly2 is a protein modification where 14-carbon saturated fatty acid myristate is covalently attached to the N-terminal Gly (exposed after removal of p.Met1) of the target protein catalyzed by N-terminal myristoyltransferases.…”

“…The lipid-modified protein is directed to a membrane by the action of N-myristoylation, but palmitoylation is required for stable anchoring to the phospholipid bilayer ( 27 ). In X-linked retinitis pigmentosa protein 2 (RP2), myristoylation alone was not sufficient to traffic amino acid residues 1 to 16 RP2 to the cilia ( 20 ). We deactivated palmitoylation on Gly2 or myristoylation on Cys3 by Gly-to-Ala or Cys-to-Ala substitutions, but observed no effect on the ciliary trafficking.…”

Lipid Modifications in Cilia Biology

“…The targeting is mediated by an N-terminal dual lipidation-coupled ciliary targeting signal (nlCTS). Palmitoylation of nlCTSs appears to be important not only for ciliary targeting, but also for the stability of the respective proteins [21].…”

“…With more protein acylation studies being carried out, it is becoming clear that acylation of ciliary proteins allows for more than just membrane attachment. Along with membrane tethering, lipid modifications are also integral for proper localization, distribution, abundance, stability and function of several cilia proteins [19,20,21,22,23,24]. The role of various lipid modifications (Table 1) on cilia protein trafficking and signaling will be discussed in the following sections (Table 2).…”