A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6, the mammalian orthologue of Chlamydomonas PF16.
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.
Zhang
Zhibing
Z
Center for Research on Reproduction and Women's Health. Department of Psychiatry. Center for Experimental Therapeutics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
Sapiro
Rossana
R
Kapfhamer
David
D
Bucan
Maja
M
Bray
Jeff
J
Chennathukuzhi
Vargheese
V
McNamara
Peter
P
Curtis
Anne
A
Zhang
Mei
M
Blanchette-Mackie
E Joan
EJ
Strauss
Jerome F
JF
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002696:Chlamydomonas / Q000378:metabolism*; D006224:Cricetinae; D005407:Flagella / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013094:Spermatozoa / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D013737:Testis / Q000737:chemistry; D014020:Tissue Extracts; D020798:Two-Hybrid System Techniques
D020418:Algal Proteins; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; C107014:PF20 protein, Chlamydomonas; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C400106:SPAG6 protein, human; D014020:Tissue Extracts