title: Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium.
abstract: Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3-cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3-cells displayed delayed streaming and aggregation, and interestingly, cln3-cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3-CM, of which 3 were absent in cln3-CM. Moreover, 12 proteins that were present in cln3-CM were absent in WT CM. Label-free quantification identified 42 proteins that were present in significantly higher amounts in cln3-CM compared to WT, and 3 proteins that were present in significantly reduced amounts. A GO term enrichment analysis showed that a majority of the affected proteins are linked to endocytosis, vesicle-mediated transport, proteolysis, and metabolism. In total, the results of this study indicate that Cln3 functions in both conventional and unconventional protein secretion and that loss of Cln3 results in deregulated secretion during early development. Importantly, this is the first evidence in any system linking CLN3 function to protein secretion.
authors.affiliation: Trent University, Department of Biology, 1600 West Bank Drive, Peterborough, Ontario, K9L 0G2, Canada. Electronic address: roberthuber@trentu.ca.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Huber
mesh_terms: D000818:Animals; D004023:Dictyostelium / Q000235:genetics; D004195:Disease Models, Animal; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009472:Neuronal Ceroid-Lipofuscinoses / Q000235:genetics* / Q000473:pathology; D010641:Phenotype; D021381:Protein Transport / Q000235:genetics; D059748:Proteolysis; D015398:Signal Transduction; D014617:Vacuoles / Q000235:genetics* / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000235:genetics
chemical_list: C105199:CLN3 protein, human; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D014909:Wheat Germ Agglutinins
keywords: Batten disease; CLN3; Development; Dictyostelium discoideum; Neuronal ceroid lipofuscinosis; Protein secretion