RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments

Abstract

Prenylation is the posttranslational modification of a carboxylterminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.protein posttranslational modification | protein transport | retinal degeneration | methyl esterification P osttranslational modifications increase a protein's functional repertoire, regulating protein-protein interactions and targeting to cellular components. One such posttranslational modification is prenylation, a three-step process that first adds a farnesyl or geranylgeranyl lipid to the C-terminal cysteine of proteins ending in a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid residue) (1-3). The next step is proteolysis of the last three amino acids (-AAX) by the protease RAS-converting enzyme 1 (RCE1) or zinc metalloproteinase sterile-24 homologue (ZMPSTE24) (4, 5). The final step is the methyl esterification of the newly exposed isoprenylcysteine residue catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT) (4).Prenylation enables peripheral membrane proteins to interact with membranes and facilitates their interaction with protein partners (2). The significance of the proteolysis and methylation steps is not entirely clear (4). In general, farnesylated proteins require postprenylation processing for proper localization, whereas the more hydrophobic geranylgeranylated proteins do not (6). Germline knockout of Rce1 or Icmt results in embryonic lethality in mice, demonstrating the importance of these processing steps during development (7,8). Subsequent conditional knockout studies revealed that proteolysis is crucial in the heart but is dispensable in liver and bone marrow (9, 10). Although Rce1 is expressed in the brain and eye, the role of RCE1-mediated endoproteolysis in neuronal tissue in vivo is not ...

ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting

“…Although it is currently unknown whether a subset of endogenous INPP5E translocates into the nucleus in response to DNA damage and is involved in the DDR pathway, interactions of INPP5E with CEP164 and other DDR-related proteins may have relevance to this pathway. Several other prenylated proteins localize to the primary cilia and the photoreceptor outer segment (e.g., RPGR, PDE6A, PDE6B, and GNGT1), and loss of these proteins or perturbation of their ciliary trafficking results in photoreceptor degeneration (12)(13)(14)(15)(16)(17)(18). Mechanisms that target these proteins to cilia and to the photoreceptor outer segment remain to be determined.…”

“…Inactivation of Inpp5e in adult mice results in obesity and photoreceptor degeneration. Interestingly, many proteins that localize to cilia, including INPP5E, RPGR, PDE6 α and β subunits, GRK1 (Rhodopsin kinase), and GNGT1 (Transducin γ chain), are prenylated (either farnesylated or geranylgeranylated), and mutations in these genes or genes involved in their prenylation (e.g., AIPL1 and RCE1) lead to photoreceptor degeneration in vertebrates and humans (12)(13)(14)(15)(16)(17)(18). Understanding the molecular mechanisms by which prenylated proteins are transported to primary cilia would have significant ramifications in developing therapeutic strategies to treat blindness and other ciliopathy phenotypes associated with these genes.…”

Identification of a Novel Prenyl and Palmitoyl Modification at the CaaX Motif of Cdc42 That Regulates RhoGDI Binding

“…PDE6␣ and PDE6␤ form an obligate heterodimer and are components of the photoreceptor cGMP phosphodiesterase. Canonical CaaX processing of PDE6␣ and -␤ is well documented (41), and it is known that RCE1 activity is required for trafficking of rod PDE6 to photoreceptor outer segments (42). Cytosolic phospholipase A2␥ undergoes classical CaaX processing when expressed in insect cells (43).…”

Targeting RAS Membrane Association: Back to the Future for Anti-RAS Drug Discovery?

“…Similarly, targeted depletion of Rce1 in the retina resulted in rapid degeneration of specific photoreceptor cells (37). On the other hand, the cleavage of RHOA by the bacterial toxin and cysteine protease YopT requires RCE1 but not ICMT (38).…”