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Molecular and Cellular Biology, November 2002, p. 7993-8004, Vol. 22, No. 22
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.22.7993-8004.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16

Zhibing Zhang,1 Rossana Sapiro,1 David Kapfhamer,2 Maja Bucan,2 Jeff Bray,1 Vargheese Chennathukuzhi,1 Peter McNamara,3 Anne Curtis,3 Mei Zhang,4 E. Joan Blanchette-Mackie,4 and Jerome F. Strauss III1*

Center for Research on Reproduction and Women's Health,1 Department of Psychiatry,2 Center for Experimental Therapeutics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104,3 Lipid Cell Biology Section, Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 208924

Received 30 May 2002/ Returned for modification 22 July 2002/ Accepted 12 August 2002

cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.

* Corresponding author. Mailing address: Center for Research on Reproduction and Women's Health, 1354 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-0147. Fax: (215) 573-5408. E-mail: jfs3{at}mail.med.upenn.edu.

Molecular and Cellular Biology, November 2002, p. 7993-8004, Vol. 22, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.22.7993-8004.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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