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

Proc. Natl. Acad. Sci. U.S.A.

Kostetskii

Moss

et al. 2004

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PF20 was first identified in Chlamydomonas rheinhardtii as an essential component of the axoneme central apparatus. We discovered that the mouse Pf20 gene encodes two major transcripts (2.5 and 1.4 kb), which are expressed in different patterns during spermatogenesis, yielding proteins of 71 and 35 kDa, respectively. Both proteins contain contiguous WD repeats in their C termini. The meiotically expressed 71-kDa protein is incorporated into the central apparatus, whereas the 35-kDa protein, which accumulates in postmeiotic male germ cells, is abundant in the nucleus. We disrupted the Pf20 gene domains that encode the C-terminal WD repeats in embryonic stem cells. Highly chimeric mice carrying the mutant Pf20 allele had impaired spermatogenesis with a significant loss of germ cells at the round spermatid stage, in association with disorganization of sperm axoneme structure. The mutated Pf20 allele was never transmitted, indicating that Pf20 haploinsufficiency caused the defects in spermatogenesis. The 35-kDa PF20 protein was shown to bind to meiosis-expressed gene 1 (MEIG1), a chromosome͞chromatin-binding protein initially expressed during meiosis but retained in the germ cell nucleus throughout later stages of spermatogenesis. Our findings reveal an essential role for Pf20 in mouse spermatogenesis, sustaining postmeiotic germ cell viability. The different patterns of expression of the two PF20 proteins suggest the possibility that the Pf20 gene has multiple functions during spermatogenesis.A xonemes, critical components of cilia and flagella, have a structure consisting of nine outer doublet microtubules encircling a central pair of microtubules, which has remained virtually unchanged during evolution (1-3). Defective assembly or function of axonemes results in immotile cilia, which causes defects in lateralization, respiratory disease, hydrocephalus, and infertility (4-7). The best-studied mutations in mammals that cause immotile cilia are in genes encoding dynein arm proteins, which generate the force that drives axonemal motility (8-10). Less is known about other axonemal proteins, especially those of the central pair. However, studies of Chlamydomonas rheinhardtii revealed important roles for the central apparatus. Mutant Chlamydomonas strains lacking functional PF16, PF6, and PF20 have paralyzed flagella, and the C1 microtubule, the projections connecting the C1 microtubule and the entire central pair, respectively, are missing in isolated axonemes (11)(12)(13).To understand the function of the central apparatus in mammals, we cloned the human and mouse orthologues of Chlamydomonas, PF16 (SPAG6) (14, 15) and PF20 (16). SPAG6-deficient mice are hydrocephalic, and males surviving to maturity are infertile as a result of a marked sperm motility defect associated with disorganization of flagellar structures, including loss of the central pair microtubules and disorganization of the outer dense fibers and fibrous sheath (6). These observations suggested an important role for central apparatus proteins in mammali...

supporting

confidence: 48%

mentioning

confidence: 99%

Haploinsufficiency for the murine orthologue of Chlamydomonas PF20 disrupts spermatogenesis

Proc. Natl. Acad. Sci. U.S.A.

Kostetskii

Moss

et al. 2004

Self Cite

“…As described previously, SPAG16L protein was also localized in the cytoplasm of SPAG16L/ pEGFP-N 2 plasmid transfected CHO cells [2], similar to the distribution pattern of TSSK2. To further study the association of the two proteins, TSSK2/DsRed-N 1 and SPAG16L/pEGFP-N 2 were co-transfected into CHO cells and the localization of each protein was examined by confocal microscopy.…”

Section: Interaction Of Spag16l and Tssk2mentioning

confidence: 69%

“…A mouse testis cDNA library was screened using the C-terminus domain of SPAG16 as the bait [2]. Plasmid DNA from the positive yeast clones was sequenced, and the candidate interacting genes were identified by searches of public databases.…”

Section: Potential Proteins That Associate With Spag16mentioning

confidence: 99%

“…Pf20 mutants have paralyzed flagella and an associated absence of the entire central pair [1]. In mammals, two SPAG16 proteins, 71 kDa SPAG16L and 35 kDa SPAG16S, are translated from a single Spag16 gene [2]. SPAG16L is translated from a 2.5 kb mRNA, and both mRNA and protein are expressed in tissues with flagellated cells or cilia, such as testis, brain and trachea [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

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Phosphorylation of Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-specific Kinase, TSSK-2.

Biology of Reproduction

Shen

Jones

et al. 2008

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Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-twohybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

Accelerated mortality from hydrocephalus and pneumonia in mice with a combined deficiency of SPAG6 and SPAG16L reveals a functional interrelationship between the two central apparatus proteins

Cell Motil. Cytoskeleton

Tang

Zhou

et al. 2007

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SPAG6 and SPAG16L are proteins localized to the "9+2" axoneme central apparatus. Both are essential for sperm motility and male fertility. These two proteins are also expressed in other tissues containing ciliated cells, such as brain and lung. To study the effects of combined deficiency of these two proteins, a double mutant mouse model was created. The double mutant mice displayed a more profound phenotype of growth retardation and hydrocephalus compared to mice nullizygous for SPAG6 and SPAG16L alone. The double mutant mice died younger, and mortality was significantly higher than in single mutant mice. In addition, the double mutant mice demonstrated pneumonia and its complications, including hemorrhage, edema, and atelectasis, phenotypes not observed in mice nullizygous for mutations in the individual genes. No other cilia-related phenotypic change was detected in double mutant mice including lateralization defects. The ultrastructure of cilia in both the brain and lung of the double mutant mice appeared normal. This model of combined SPAG6 and SPAG16L deficiency provides a new platform to study primary ciliary dyskinesia. The findings also demonstrate that SPAG6 and SPAG16L have related roles in controlling the function of cilia in the brain and lung.