Localization and function of nephrocystin-4 in Chlamydomonas

Junya Awata1, Saeko Takada1, Karl F. Lechtreck1, Eric Johnson1, Bethany L. Walker1, Gregory J. Pazour2, Robert A. Bloodgood3, and George B. Witman1

1) Cell Biology and 2) Molecular Medicine, U. Mass. Med. School, Worcester MA 01655 USA 3) Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908 USA

Mutations in human NPHP4, encoding nephrocystin-4, cause nephronophthisis (kidney disease) and retinitis pigmentosa (blindness). It has been reported that the Caenorhabditis elegans homologue of nephrocystin-4 is specifically located in the transition zone of cilia. Using immunofluorescence microscopy with antibodies against the Chlamydomonas homologue of nephrocystin-4, we observed that the Chlamydomonas protein likewise is located in the proximal region of the flagellum. Furthermore, immuno-gold electron microscopy of Chlamydomonas cells expressing HA-tagged nephrocystin-4 and probed with antibodies against the HA peptide indicated that nephrocystin-4-HA is specifically localized to the transition zone. Therefore, the location of nephrocystin-4 is conserved among species, suggesting that its function also has been conserved. We have identified a Chlamydomonas insertional mutant in which the nephrocystin-4 gene is completely deleted, and are using it to investigate t he function of nephrocystin-4. The mutant flagella have normal length and motility, but cells swim in paths less straight than those of wild type. The phenotype is rescued by transformation with a wild-type Chlamydomonas nephrocystin-4 gene. Electron microscopy indicates that the mutants have abnormal transition zones, apparently normal axonemes, and a reduced flagellar membrane glycocalyx. Immunofluorescence microscopy and western blotting revealed that the amount of the major flagellar membrane glycoprotein FMG-1B is reduced in the mutant flagella. However, the amounts of other membrane proteins, specifically PKD2 and the mastigoneme protein, are normal. The results suggest that nephrocystin-4 has a role in moving a subset of membrane proteins into the flagellum.

e-mail address of presenting author: Junya.Awata@umassmed.edu