Regulated processing and secretion of a peptide precursor in cilia

Abstract

Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia-derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Peptidergic intercellular communication controls a wide range of physiological and behavioral responses and occurs throughout eukaryotes. The Chlamydomonas reinhardtii genome encodes what appear to be numerous prepropeptides and enzymes homologous to those used to convert metazoan prepropeptides into bioactive peptide products. Since C. reinhardtii , a green alga, lack the dense core vesicles in which metazoan peptides are processed and stored, we explored the hypothesis that propeptide processing and secretion occur through the regulated release of ciliary ectosomes. A synthetic peptide (GATI- amide ) that could be generated from a 91-kDa peptide precursor (proGATI) serves as a chemotactic modulator, attracting minus gametes while repelling plus gametes. Here we dissect the processing pathway that leads to formation of an amidated peptidergic sexual signal specifically on the ciliary ectosomes of plus gametes. Unlike metazoan propeptides, modeling studies identified stable domains in proGATI. Mass spectrometric analysis of a potential prohormone convertase and the amidated proGATI-derived products found in cilia and mating ectosomes link endoproteolytic cleavage to ectosome entry. Extensive posttranslational modification of proGATI confers stability to its amidated product. Analysis of this pathway affords insight into the evolution of peptidergic signaling; this will facilitate studies of the secretory functions of metazoan cilia.

Cilia Provide a Platform for the Generation, Regulated Secretion, and Reception of Peptidergic Signals

“…Furthermore, bioactive PAM protein and its amidated products have been identified in ciliary ectosomes released during sexual reproduction in C. reinhardtii, suggesting an ancient functional connection between cilia and amidated peptide release [5]. Indeed, biochemical studies of one identified amidated product (termed GATI-amide) in C. reinhardtii revealed that the amidated propeptide precursor was processed to the final amidated product on the ciliary membrane likely during trafficking into nascent ectosomes ( [28]; this pathway is discussed in detail below.…”

“…Biochemical tools enable the processing of the large (nominally 91 kDa) proGATI precursor and the synthesis of the amidated bioactive product to be followed. In mammals, cell-type-specific subtilisinlike prohormone convertases are involved in the processing of propeptide precursors; C. reinhardtii utilizes the subtilisin-like protease VLE1 as a ciliary prohormone convertase to cleave proGATI [28]. This suggests that C. reinhardtii uses cilia and ciliary ectosomes as a platform for peptide processing and secretion.…”

“…This suggests that C. reinhardtii uses cilia and ciliary ectosomes as a platform for peptide processing and secretion. Indeed, ciliary ectosomes were found to carry multiple peptide products generated from a single propeptide precursor that potentially perform diverse cellular functions [28].…”

“…Importantly, both PAM and amidated products localize to cilia and are secreted into the environment on ciliary ectosomes. For example, in C. reinhardtii, an amidated product (GATI-amide) that is synthesized from a large proGATI precursor and secreted from cilia acts as a chemotactic modulator, ultimately controlling the ciliary motility of gametic cells [5,27,28].…”

Extracellular vesicles highlight many cases of photoreceptor degeneration

“…Another specialized plasma membrane structure, the primary cilium, has attracted significant interest for its ability to release ectosomes [recent reviews: ( Wang and Barr, 2018 ; Luxmi and King, 2022 ; Ojeda Naharros and Nachury, 2022 ; Vinay and Belleannee, 2022 )]. Some of the functions ascribed to ciliary ectosomes include selectively discarding proteins from the cilium ( Nager et al, 2017 ), facilitating cell cycle reentry ( Phua et al, 2017 ), and releasing bioactive proteins and peptides ( Wood et al, 2013 ; Wang et al, 2014 ; Luxmi et al, 2022 ; Walsh et al, 2022 ). Although precise molecular mechanisms driving ciliary ectosome release are unknown, the process has been shown to rely on the actin cytoskeleton in cultured cells ( Nager et al, 2017 ; Phua et al, 2017 ) and ESCRT in Chlamydomonas ( Long et al, 2016 ).…”