The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown—including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation—suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.

Author Summary

ADPKD, the most common potentially lethal monogenetic disorder, is caused by mutations in PKD1 and PKD2. We are beginning to appreciate the important roles these gene products, and others, play in cilia, which are thin rod-like organelles projecting from the cell surface. Defects in cilia function are associated with a variety of human diseases, including all variants of polycystic kidney disease. Despite intense study of cilia and how they influence disease, it is not understood how proteins are targeted and delivered to cilia. Our work provides the first link between the exocyst, a conserved eight-protein complex involved in protein localization, and a disease gene, PKD2. Knockdown of the exocyst protein Sec10 results in a number of cellular- and cilia-related phenotypes that are also seen upon pkd2 loss—both in kidney cells and zebrafish. We then demonstrate specific genetic and biochemical interactions between sec10 and pkd2. We further show that Sec10 interacts with other ciliary proteins, such as IFT20 and IFT88. From this work, we propose that the exocyst is involved in bringing multiple types of ciliary proteins to the cilium. Given that the exocyst is required for cilia structure and function, the exocyst may play a role in cilia-related human diseases.