Cystin localizes to primary cilia via membrane microdomains and a targeting motif.
Primary cilia are dynamic, complex structures that contain >500 proteins, including several related to polycystic kidney disease. How these proteins target to cilia and assemble is unknown. We previously identified Cys1 as the gene responsible for disease in Cys1(cpk) mice, a mouse model of autosomal recessive polycystic kidney disease; this gene encodes cystin, a 145-amino acid cilium-associated protein. Here, we characterized the localization of cystin in the embryonic kidney and liver, in isolated renal collecting ducts, and in an inner medullary collecting duct mouse cell line. Because endogenous levels of cystin expression are low, we generated inner medullary collecting duct cell lines that stably express enhanced green fluorescence protein-tagged constructs of wild-type cystin or various truncation mutants. We determined that cystin is myristoylated at its G2 residue and that N-myristoylated cystin fractionates with membrane microdomains. Furthermore, the N-myristoylation signal is necessary but not sufficient to target cystin to the primary cilium. Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease.
Departments of Medicine and Genetics, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL 35294, USA.
Binli
B
Tao
Su
S
Bu
Zhihua
Z
Yang
Brian
B
Siroky
John C
JC
Kappes
Andreas
A
Kispert
Lisa M
LM
Guay-Woodford
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002923:Cilia / Q000378:metabolism*; D004195:Disease Models, Animal; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016891:Polycystic Kidney, Autosomal Dominant / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C482294:CYS1 protein, human; C449819:Cys1 protein, mouse; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins