Drosophila melanogaster Importin 1 and 3 Can Replace Importin 2 During Spermatogenesis but Not Oogenesis

Corresponding author: David S. Goldfarb, University of Rochester, Rochester, NY 14627., dasg{at}mail.rochester.edu (E-mail)

Communicating editor: K. V. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Importin 's mediate the nuclear transport of many classical nuclear localization signal (cNLS)-containing proteins. Multicellular animals contain multiple importin genes, most of which fall into three conventional phylogenetic clades, here designated 1, 2, and 3. Using degenerate PCR we cloned Drosophila melanogaster importin 1, 2, and 3 genes, demonstrating that the complete conventional importin gene family arose prior to the split between invertebrates and vertebrates. We have begun to analyze the genetic interactions among conventional importin genes by studying their capacity to rescue the male and female sterility of importin 2 null flies. The sterility of 2 null males was rescued to similar extents by importin 1, 2, and 3 transgenes, suggesting that all three conventional importin 's are capable of performing the important role of importin 2 during spermatogenesis. In contrast, sterility of 2 null females was rescued only by importin 2 transgenes, suggesting that it plays a paralog-specific role in oogenesis. Female infertility was also rescued by a mutant importin 2 transgene lacking a site that is normally phosphorylated in ovaries. These rescue experiments suggest that male and female gametogenesis have distinct requirements for importin 2.

THE nuclear targeting of proteins is mediated by a number of nuclear localization signal (NLS)-specific receptors called importins or karyopherins (MATTAJ and ENGLMEIER 1998 ; WEIS 1998 ; GORLICH and KUTAY 1999 ; NAKIELNY and DREYFUSS 1999 ). Importin ß-family members bind NLS cargo in the cytoplasm and act as chaperones to facilitate their translocation across the nuclear envelope through serial interactions with nucleoporins arrayed along the central channel of the nuclear pore complex (ROUT et al. 2000 ; RABUT and ELLENBURG 2001 ). Importin ß1 is unusual in that it functions in conjunction with importin to mediate the import of "classical" NLS (cNLS) cargo (NAKIELNY and DREYFUSS 1999 ), which includes a large variety of nuclear proteins (MICHAUD and GOLDFARB 1993 ).

Importin 's are composed of 10 tandem armadillo (Arm) repeats, bracketed by shorter N- and C-terminal domains (PEIFER et al. 1994 ; CONTI et al. 1998 ). Interestingly, importin ß-family members contain large domains composed of tandem HEAT motifs, which are related to Arm repeats by a degenerate 40- to 45-amino-acid consensus sequence (MALIK et al. 1997 ; ANDRADE et al. 2001 ). Arm and HEAT repeats fold into structurally related superhelical rods that serve as selective scaffolds for binding proteins (HUBER et al. 1997 ; CONTI et al. 1998 ). Importin 's bind cNLS cargo via their Arm domains (CONTI et al. 1998 ) and importin ß through N-terminal importin ß-binding (IBB) sequences (GORLICH et al. 1996 ; WEIS et al. 1996 ). After cNLS-importin /ß1 ternary complexes enter and dissociate in the nucleus, importin and ß1 are independently recycled back to the cytoplasm (KOEPP et al. 1996 ; PERCIPALLE et al. 1997 ). Export of importin is mediated by another importin ß-family member, an exportin called Cse1p in yeast and CAS in higher eukaryotes (KUTAY et al. 1997 ; HOOD and SILVER 1998 ), which binds within the tenth Arm repeat of importin (HEROLD et al. 1998 ).

In contrast to the single importin gene of Saccharomyces cerevisiae (SRP1), vertebrates contain as many as eight conventional importin genes. Phylogenetic analysis has revealed that the majority of importin genes belong to one of three conserved clades (KOHLER et al. 1997 , KOHLER et al. 1999 ; MALIK et al. 1997 ), referred to here as 1, 2, and 3. All conventional yeast and plant importin genes are 1 paralogs. In contrast, metazoan animals typically contain representatives of all three clades. The functional basis for the conservation of multiple importin genes in animals is not known. However, their occurrence only in metazoan animals suggests they may be involved in tissue differentiation and development. Consistent with this notion, importin 1's, 2's, and 3's show distinct tissue and cell type-specific expression patterns (PRIEVE et al. 1996 ; KOHLER et al. 1997 ; TSUJI et al. 1997 ; NACHURY et al. 1998 ; KAMEI et al. 1999 ).

In vitro evidence suggests that all conventional importin 's bind a broad class of cNLS sequences, but they do so with different affinities (PRIEVE et al. 1996 , PRIEVE et al. 1998 ; NADLER et al. 1997 ; MIYAMOTO et al. 1997 ). In permeabilized cell nuclear import assays, representative importin paralogs show overlapping preferences for different cNLS sequences (KOHLER et al. 1999 ). In some cases, specific importin 's show strong preferences for NLS cargo. For example, only an importin 3 mediates the in vitro import of RCC1 (KOHLER et al. 1999 ) and Ran BP3 (WELCH et al. 1999 ), and only an importin 1 imports the Stat 1 transcription factor (SEKIMOTO et al. 1997 ). In addition, the preference of importin 's for certain NLS cargo is significantly increased when two different substrates are presented together in the import assay (KOHLER et al. 1999 ). This latter finding underscores the complexity of the functional interactions between importin 's and different NLS cargo and indicates that in vivo studies are needed to unravel the physiological roles of the importin 1, 2, and 3 genes. In this study we exploit the finding that Drosophila importin 2 is required for male and female fertility to examine functional interactions among the conserved Drosophila importin genes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cloning of Drosophila importin genes and phylogenetic analysis:
Drosophila importin genes were cloned using degenerate oligonucleotide-mediated PCR. The 5' oligonucleotide was made to the conserved amino acid sequence AWALTNIA found in the third helix of arm repeat 2. To facilitate cloning, an EcoRI site was added, generating the sequence 5' ATCGCGAATTCGC/TITGGGCIT/CTIACIAAT/CATT/C/AGC 3' (I represents inosine). The 3' oligonucleotide was made to the conserved VGNIVTG sequence in the third helix of arm repeat 6. With the addition of a BamHI site, the nucleotide sequence of the 3' primer is 5' GACGTAGGATCCCCIGTTACT/G/AATG/ATTICCIA 3'. The primers were used to PCR amplify importin gene fragments [PCR conditions: 1.5 mM MgCl₂, 20 mM Tris-Cl pH 8.4, 50 mM KCl, 0.2 µg SalI-digested Drosophila genomic DNA, 2 µM 5' and 3' primers, 200 µM dNTPs, and 1 unit of Taq DNA polymerase (Bethesda Research Laboratories, Gaithersburg, MD) amplified at 94° for 20 sec, 43° for 20 sec, ramp to 55° for 2 min, 55° for 30 sec, 72° for 30 sec with 50 cycles] from genomic Drosophila DNA. Genomic DNA was digested with SalI to inhibit importin 2 amplification since PCR with undigested DNA yielded only importin 2 products. Two distinct PCR products were subcloned into the pGAD 424 vector (CLONTECH, Palo Alto, CA) and sequenced to reveal that they encoded novel importin 1 and 3 genes. The two importin gene fragments were Digoxigenin labeled by PCR (Boehringer Mannheim, Indianapolis) and used to probe an ovary gt11 cDNA library (ZINN et al. 1988 ) to obtain full-length coding regions (1, accession no. AAC26055; 3, accession no. AAC26056). A CLUSTAL V (HIGGINS et al. 1992 ) alignment of the new Drosophila importin genes to the importin -like genes found in GenBank was used to construct a phylogenetic tree by the neighbor-joining method (SAITOU and NEI 1987 ).

Genetic stocks and markers:
Flies were kept on standard cornmeal-dextrose media and grown at 25°. The importin 2 gene was previously referred to as the oho31 gene and encodes the Pendulin protein (KUSSEL and FRASCH 1995 ; TOROK et al. 1995 ). The clean importin 2^D14/y⁺ CyO and importin 2^D3/y⁺ CyO stocks were provided by Dr. Istvan Kiss (Hungarian Academy of Sciences, Szeged, Hungary; see TOROK et al. 1995 ). Deficiency stocks Df(3L)kto2/TM6B, Tb[1], Df(3R)by416/TM3, Sb[1], Gal4^Hsp70/TM6B, and w¹¹¹⁸ stocks were obtained from the Bloomington Stock Center. The Gal4^nanos-VP16 stock (RORTH 1998 ) was a gift from Pernille Rørth (EMBL, Heidelberg, Germany).

Fertility assays and testes squashes:
Male fertility was assayed by crossing individual males to five w¹¹¹⁸ virgin females. Female fertility was assayed by mating individual females to three w¹¹¹⁸ males. Flies were allowed to mate for 10 days before being discarded. To examine sperm motility, importin 2^D14 or importin 2 ^D3 homozygous and heterozygous testes were dissected from 4-day-old males in cold testis dissecting buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris-HCl pH 6.8), gently ruptured under a coverslip, and visualized by dark field microscopy.

Transmission electron microscopy of testes:
Testes were dissected in cold testis dissecting buffer from importin 2^D14/importin 2^D14, importin 2^D14/y⁺CyO, and w¹¹¹⁸ 0- to 4-day-old males and prepared for electron microscopy as previously described (TOKUYASU et al. 1977 ). Briefly, samples were fixed in 2.5% glutaraldehyde, postfixed in 1.0% osmium tetroxide, infiltrated in Spurr epoxy resin, and embedded. These samples were then cut into ultra-thin sections, stained with uranyl acetate and lead citrate, and subsequently examined with a Hitachi 7100 transmission electron microscope.

RNA and protein isolation:
Total RNA and proteins were isolated from Drosophila tissues with Tri-Reagent LS (Molecular Research Center, Cincinnati; CHOMCZYNSKI 1993 ) following the recommended protocols. RNA was quantified by determining the A260, and protein concentration was determined using the Bio-Rad (Richmond, CA) DC protein assay.

Northern and Western blots:
To determine the endogenous importin 1, 2, and 3 mRNA expression patterns, 20 µg of RNA isolated from adult males, adult females, dissected testes, and dissected ovaries was separated on a 1% agarose, 6% formaldehyde gel; transferred to a nylon membrane; and probed with ³²P-random prime-labeled importin 1, 2, or 3 probes [GIBCO (Grand Island, NY)/Bethesda Research Laboratories random primers DNA labeling system]. Bands were visualized by phosphoimaging. To examine protein expression patterns, 10 µg of protein isolated from adult males, adult females, dissected testes, and dissected ovaries was separated on an 8% PAGE gel; transferred to a polyvinylidene fluoride membrane; and blotted with rabbit anti-importin 2 (TOROK et al. 1995 ) provided by Istvan Török (DKFZ, Heidelberg, Germany) or rabbit anti-importin 3 (MATHE et al. 2000 ) provided by Endre Máthé (University of Cambridge, Cambridge, United Kingdom). Blots were developed using alkaline phosophatase-tagged goat anti-rabbit secondary antibodies. To quantify transgene expression levels RNA and protein were isolated from dissected ovaries from mated w¹¹¹⁸ or homozygous importin 2^D14 females expressing UASp importin 1, 2, 3, 2x 3, or 2S56A transgenes (see next section). Ovarian RNA (15 µg) was tested in a Northern blot using ³²P-random prime-labeled K10 3' untranslated region (UTR) and RP49 probes as described above. Bands were visualized and quantified by phosphoimaging. To examine protein levels 10 µg of protein was tested in a Western blot using rabbit anti-importin 2, rabbit anti-importin 3, or mouse anti--tubulin antibodies (Amersham, Arlington Heights, IL) as described above.

Immunofluorescence of testes:
Testes were dissected from wild-type or homozygous importin 2^D14 males in 1x PBS; fixed in 1x PBS, 4% paraformaldehyde; and blocked in PBS-saponin (1x PBS, 0.2% saponin, and 0.3% normal goat serum). Testes were then incubated with rabbit anti-importin 2 (TOROK et al. 1995 ) diluted 1:50 in PBS-saponin, followed by a goat anti-rabbit FITC-labeled secondary antibody diluted 1:300 in PBS-saponin. DNA was stained with 10 µM Hoechst in PBS. Confocal microscopy was performed on a Leica TCS NT microscope equipped with UV, Ar, Kr/Ar, and He/Ne lasers, and digital images were processed using Adobe PhotoShop (Adobe Systems, San Jose, CA).

Expression constructs and germline transformations:
Importin transgenic flies were created by cloning importin 1, 2, and 3 PCR fragments corresponding to the 1.5-kb coding region of each gene into EcoRI and NotI sites in the pUASt P-element transformation vector (BRAND and PERRIMON 1993 ) or into KpnI and NotI sites in the UASp P-element transformation vector (RORTH 1998 ). UASp 3 contains an additional 22 nucleotides of its 5' UTR. The UASp importin 2 S56A transgene was created by PCR amplifying an importin 2 3' AvaII and NotI fragment containing a single point mutation that changed the TCG codon for serine-56 to a GCG alanine codon. This PCR fragment was then ligated with a 5' KpnI and AvaII importin 2 PCR fragment into KpnI and NotI sites in UASp. The pUAS_t and UASp importin 2 and 3 transgenes contain an additional 7 and 42 nucleotides of their 3' UTRs, respectively, and UASp importin 3 contains 22 nucleotides of its 5' UTR.

Transgenic UASt and UASp importin 1, 2, 3, and UASp importin 2 S56A lines were created using standard germline transformation procedures (SPRADLING 1986 ). The UASt and UASp importin 1 and UASp importin 2 inserts used in this study were located on the second chromosome, while the UASt importin 2, UASt importin 3, UASp importin 3, and UASp importin 2 S56A inserts map to the third chromosome.

Crosses:
To examine genetic interactions between the importin 2^D14 allele and deficiencies that uncover the 1 or 3 genes, females of the genotype importin 2^D14/CyO were crossed to (1) importin 1^Df(3L)kto2/TM6B or (2) importin 3^{Df(3R) by416}/TM3. Male offspring without balancers are heterozygous for importin 2 and importin 1 or 3. CyO males without TM3 or TM6B balancers serve as heterozygous importin 1 or 3 flies in a wild-type importin 2 background. To test for rescue of importin 2^D14 male sterility following ectopic importin 1, 2, or 3 expression, females of the genotype importin 2^D14/CyO; Gal4^Hsp70/TM6B were crossed to (1) UASt importin 1, importin 2^D14/CyO; (2) importin 2^D14/CyO; UASt importin 2/UASt importin 2; or (3) importin 2^D14/CyO; UASt importin 3/UASt importin 3. Male offspring that are heterozygous for the importin 2^D14 allele and have the Gal4^Hsp70 driver and the UASt importin transgenes were used as the positive control for fertility (e.g., importin 2^D14/CyO; UASt importin 2/Gal4^Hsp70); homozygous importin 2^D14 males that have the Gal4^Hsp70 driver and the UASt importin transgenes served as the experimental group (e.g., importin 2^D14/importin 2^D14; UASt importin 2/Gal4^Hsp70); homozygous mutant males that contain the UASt importin transgene but inherited the TM6B balancer instead of Gal4^Hsp70 were utilized as the negative control (e.g., importin 2^D14/importin 2^D14; UASt importin 2/TM6B). Male flies were collected 0–18 hr after eclosion, heat shocked for 2 hr in a 37° air incubator, and then assayed for fertility by mating individually to five w¹¹¹⁸ virgin females at 25°. To test for rescue of importin 2^D14 female sterility following ectopic importin 1, 2, 3, or 2 S56A expression, females of the genotype importin 2^D14/CyO; Gal4^nanos-VP16/TM6B were crossed to (1) UASp importin 1, importin 2^D14/CyO; (2) UASp importin 2, importin 2^D14/CyO; (3) importin 2^D14/CyO; UASp importin 3/ UASp importin 3; or (4) importin 2^D14/CyO; UASp importin 2 S56A/TM6B. Female offspring that are heterozygous for the importin 2^D14 allele and have the Gal4^nanos-VP16 driver and the UASp importin transgenes were used as the positive control for fertility (e.g., importin 2^D14, UASp importin 2/CyO; Gal4^nanos-VP16/+); homozygous importin 2^D14 females that have the Gal4^nanos-VP16 driver and the UASp importin transgenes served as the experimental group (e.g., importin 2^D14/importin 2^D14, UASp importin 2; Gal4^nanos-VP16/+); homozygous mutant females that contain the UASp importin transgene but inherited the TM6B balancer instead of Gal4^nanos-VP16 were utilized as the negative control (e.g., importin 2^D14, UASp importin 2/importin 2^D14; TM6B/+). To express two copies of importin 3 in an 2 mutant background importin 2^D14/CyO;/UASp importin 3, Gal4^nanos-VP16/TM6B females were crossed to importin 2^D14/CyO; UASp importin 3/UASp importin 3 and female offspring of the genotype importin 2^D14/importin 2^D14; UASp importin 3, Gal4^nanos-VP16/UASp importin 3 were collected. Females of the described genotypes were assayed for fertility as described above or used as a source for RNA and protein isolation from ovaries.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Phylogenetic structure of the Drosophila importin gene family:
Phylogenetic analysis of known importin gene sequences indicates that metazoan animals contain at least one representative of each of three conserved importin clades, designated here as conventional 1, 2, and 3 genes (Fig 1; MALIK et al. 1997 ; KOHLER et al. 1997 , KOHLER et al. 1999 ). For example, the current version of the human genome encodes four importin 1, one importin 2, and three importin 3 genes (VENTER et al. 2001 ). Of these three clades, only importin 1 genes have been found in fungi and plant genomes. Nonconventional importin genes, which contain recognizable IBB domains and conserved cNLS-binding sites, occur in plants, invertebrates, and protozoa. These divergent importin genes probably arose from the same progenitors that gave rise to extant conventional genes. At the time this project was initiated, Pendulin, an importin 2, was the only known Drosophila importin (KUSSEL and FRASCH 1995 ; TOROK et al. 1995 ).

View larger version (38K): In this window In a new window Download PPT slide   Figure 1. A phylogeny of the importin 's. Alignments of importin protein sequences were used to construct a phylogeny (MATERIALS AND METHODS). Shown here is the consensus tree of those importin sequences whose Arm domains are demonstrably homologous (see text). Bootstrap values are represented as a percentage of 1000 replications. Species, protein identification (PID) numbers, and proposed 1, 2, and 3 nomenclature for conventional genes are indicated. Vertebrate genes are shown in brown, invertebrates in red, plants in green, and fungi in blue.

Prior to the completion of the Drosophila genome sequence, we attempted to clone by PCR the entire Drosophila importin gene family using degenerate oligonucleotide primers designed against sequences that were well conserved among known importin genes. Successfully amplified PCR products were then used to screen a Drosophila ovary cDNA library for full-length importin cDNAs. In addition to recloning the Drosophila importin 2, this strategy yielded novel importin 1 and 3 genes. The new importin genes encode Arm domain proteins with consensus importin ß and cNLS binding domains. These results demonstrate that the phylogenetic structure of the importin gene family is conserved between invertebrates and vertebrates (see also KOHLER et al. 1999 ). The Drosophila importin 3 gene was recently recloned by two hybrid screens on the basis of its interactions with DNA polymerase (MATHE et al. 2000 ) and germ cell-less (DOCKENDORFF et al. 1999 ). In addition, the published Drosophila genome sequence (ADAMS et al. 2000 ) contains the importin 1, 2, and 3 genes, as well as a fourth nonconventional importin gene [protein identification (PID) no. g7295403] that may or may not function in cNLS transport.

The phylogeny shown in Fig 1 does not contain all importin -like sequences found in GenBank. Because importin 's contain tandem Arm repeats, sequence alignments cannot be used in constructing a rigorous phylogeny unless it can be determined that the order of individual repeats has been conserved (MALIK et al. 1997 ). If, for example, shuffling, substitution, or deletion of repeats within an Arm domain had occurred, then the alignment of this altered sequence with other sequences would effectively be comparing nonhomologous sequences. All putative importin sequences included in the phylogeny shown in Fig 1, including the nonconventional genes, contain virtually the same order of individual Arm repeats, although Arm repeat one is naturally extremely divergent. The Caenorhabditis elegans genome contains three putative importin genes, only two of which pass this homology test and are included on the phylogeny. The Arm repeats of the other C. elegans importin -like gene are dissimilar to other known importin -like sequences and cannot, therefore, be aligned with confidence, even though it is a functional importin (GELES and ADAM 2001 ). This is basically the reason that ß-catenins, plakoglobins, the flagellar protein PF16, and other non-importin Arm domain proteins that share relatively high sequence similarity with importin 's are also left off this tree. The nonconventional Oryza sativa (rice) and Arabidopsis thaliana importin sequences are included in the phylogeny because 6 of their 10 Arm repeats are homologous with those of bona fide conventional genes. Interestingly, the nonconventional O. sativa and A. thaliana importin genes define a distinct clade that arose at least 75 million years ago, prior to the division between monocotyledonous (sativa) and dicotyledonous (thaliana) angiosperms.

On the basis of the robust distribution of most known importin genes into three distinct clades (Fig 1), we propose that the 1, 2, 3 nomenclature be adopted for naming conventional importin genes. This approach clarifies nonsystematic schemes (e.g., Qip1, RCH1, etc.) and has the advantage over the previously proposed "S, P, Q" system (TSUJI et al. 1997 ) in that the numerical identifier is incorporated directly into the importin name (e.g., importin 1). This scheme is not meant to replace the original names given by their discoverers. Rather, as we learn more about the physiological role(s) of individual genes it will be helpful for comparative purposes to know that human Qip1, for example, is an importin 3.

Drosophila importin 2 is required for male and female fertility:
Two early studies reported that flies homozygous for a null allele of the importin 2 gene (oho-31) die during development while exhibiting an overgrowth of hematopoietic tissues (KUSSEL and FRASCH 1995 ; TOROK et al. 1995 ). However, these phenotypes were subsequently found to be due to a second site mutation (I. KISS, personal communication). Bona fide importin 2 null alleles, importin 2^D14 and importin 2^D3, generated by imprecise P-element mobilization (TOROK et al. 1995 ) and subsequent recombination to remove the oho31 mutation, were kindly provided to us by I. Kiss (Hungarian Academy of Sciences, Szeged, Hungary). These deletions removed 1.7 kb of 5' sequence in importin 2^D14 and 1 kb of 5' sequence in importin 2^D3 (TOROK et al. 1995 ). The large amount of coding sequence removed and the absence of importin 2 mRNA (not shown) or protein (TOROK et al. 1995 ) in homozygous importin 2^D14 flies demonstrate that these mutations are indeed protein null alleles. Consistent with the unpublished observation that the oho31 phenotype was caused by a second site mutation (I. KISS, personal communication), we do not observe any larval lethality or overgrowth of hematopoietic tissues in flies homozygous for these alleles, despite the fact that Importin 2 protein is absent. Because the importin 2^D14 and importin 2^D3 alleles are indistinguishable phenotypically (not shown), we used only the importin 2^D14 allele in our subsequent experiments.

Males homozygous for importin 2^D14 were mostly sterile but were otherwise morphologically wild type. Male fertility was quantified by mating individual male flies with five wild-type (w¹¹¹⁸) virgin females. In this experiment, all 20 heterozygous importin 2^D14 males tested were fertile, and all 20 homozygous importin 2^D14 males were sterile (Table 1). However, in rare cases, homozygous importin 2^D14 males produced a few offspring when mated with wild-type females (Table 2). Therefore, importin 2 is important but not essential for male fertility. Likewise, homozygous importin 2^D14 adult females appear morphologically wild type but are sterile (G. ADAM and I. KISS, unpublished results). We independently quantified female fertility by individually mating 20 homozygous importin 2^D14 (2^-/-) or 20 heterozygous importin 2^D14 (2^+/-) females with three wild-type males. All 2^+/- females were fertile and all 2^-/- mutant females were sterile (Table 1). In addition, we have repeatedly mated 2^-/- females with wild-type and 2^+/- males during the course of our studies and have never observed any fertile females. Therefore, importin 2 appears to be essential for female fertility but only important for male fertility.

View this table: In this window In a new window   Table 1. Fertility of importin -deficient flies

View this table: In this window In a new window   Table 2. Rescue of importin 2 null male and female sterility

Importin 2 is required for normal gametogenesis in males and females:
Because 2^-/- males and females develop and behave indistinguishably from their heterozygous 2^+/- siblings, we focused on the role of importin 2 in gametogenesis. Intact testes isolated from 2^-/- males appeared normal under the light microscope and contained characteristically large numbers of elongated sperm bundles. However, squashed testes from 4- to 5-day-old 2^-/- males released few, if any, motile sperm (data not shown). In contrast, swarms of motile sperm were released from squashed testes isolated from wild-type and 2^+/- males. In addition, no sperm were observed in the seminal receptacle or spermathecum of wild-type females mated to 2^-/- males (data not shown). Therefore, it is likely that the infertility of males lacking importin 2 is due to a defect during spermatogenesis that results in defective sperm. The occasional fertility of 2^-/- males is consistent with the occurrence of small numbers of motile sperm in some testes. An analysis of mutant testes by transmission electron microscopy (TEM) indicated that 2^-/- males display a defect in spermatogenesis upon individualization. Prior to individualization, the sperm bundles of homozygous 2^-/- flies were indistinguishable from bundles in heterozygous testes (not shown). Following individualization, most sperm bundles from heterozygous testes contained close to the full complement of 64 properly individualized spermatids (Fig 2A). In contrast, in 2^-/- testes a large number of spermatids failed to individualize and remained syncytial (Fig 2B and Fig C). This phenotype is not likely to be caused by a defect in the individualization machinery since normal-appearing individualization complexes, visualized with rhodamine-phallodin staining (FABRIZIO et al. 1998 ), were observed in 2^-/- testes (not shown). We were curious about the axonemal structure of 2^-/- sperm tails, partly because mutants in a Chlamydomonas importin -like flagellar protein, PF16, are paralyzed, and detergent-treated mutant axonemes lack central C1 microtubules. PF16 is conserved between Giardia lamblia and mice, but appears to be absent from the Drosophila genome (ADAMS et al. 2000 ). Thus it is possible that an importin replaces the function of PF16 in Drosophila flagella. However, the organization of the sperm axonemes and the structure of the major and minor mitochondrial derivatives were normal in 2^-/- testes (Fig 2). We conclude that Drosophila Importin 2 probably does not serve a comparable function to PF16 in flagella.

View larger version (138K): In this window In a new window Download PPT slide   Figure 2. Ultrastructure of importin 2+/- and 2-/- testes. (A) Electron microscopic images of mature sperm bundles of 2+/- males showing uniformly packed and individualized (arrowheads) spermatids; (B and C) 2-/- testes showing both individualized (arrowheads) and unindividualized syncytial spermatids (arrows). (D) A detail of individualized 2+/- sperm tails and (E) 2-/- sperm tails. The axoneme shown in E lacks a membrane (compare to D) and has not, therefore, completed individualization.

In our hands, the eggs laid by 2^-/- females showed gross morphological defects that corroborate earlier observations (G. ADAM and I. KISS, personal communication). Specifically, eggs laid by 2^-/- mothers were smaller and appeared deflated as if they were deficient in internal contents (not shown). In addition, dorsal appendages were missing, fused together, or otherwise severely deformed, and the chorions appeared thinner than in eggs laid by wild-type females (not shown). These results suggest that the complete sterility of 2^-/- mutant female flies is due to a severe defect in oogenesis.

Importin 1, 2, and 3 are expressed in testes and ovaries:
The results described above indicate that importin 2 is required for normal gametogenesis. Northern blot analysis was used to begin to assess the particular roles of the three conventional importin genes in gametogenesis. As shown in Fig 3A, importin 1, 2, and 3 mRNAs were all detected in testes, ovaries, and in adult male and female flies. Therefore, as is the case with the three characterized C. elegans importin genes (GELES and ADAM 2001 ), the three conventional Drosophila Importin genes are all expressed in the germline. Importin 3 mRNA levels were about equally expressed in ovaries and testes. In contrast, importin 1 mRNA levels were slightly elevated in the testes compared to ovaries, and importin 2 mRNA levels were much higher in the testes than ovaries. Thus importin 2 and, to a lesser extent, importin 1 mRNAs are selectively enriched in testes.

View larger version (65K): In this window In a new window Download PPT slide   Figure 3. Analysis of importin gene expression. (A) A total of 20 µg of total RNA isolated from adult wild-type males, adult wild-type females, dissected wild-type testes (Test.), and dissected wild-type ovaries (Ova.) was hybridized with importin 1, 2, or 3-specific probes (MATERIALS AND METHODS). Importin 1, 2, and 3 mRNA migrate at 2.5 kb. (B) A total of 10 µg of total protein isolated from adult wild-type males, adult wild-type females, dissected wild-type testes (Test.), and dissected wild-type ovaries (Ova.) was examined on a Western blot with rabbit anti-Importin 2 or 3 antibodies (MATERIALS AND METHODS).

Western blot analyses of Importin 2 and 3 protein levels are shown in Fig 3B. Importin 1 protein levels could not be examined because antibodies are not currently available. The concentration of Importin 3 protein is about the same in testes and ovaries, but somewhat higher in adult females than in males (see also MATHE et al. 2000 ). This result is consistent with the Northern blot analysis (Fig 3A), which shows similar importin 3 mRNA levels in adult and germ cell extracts. Thus, Importin 3 protein levels appear to be uniformly expressed in somatic and germline tissues in proportion to its mRNA levels. The Importin 2 case is complex. Here, Importin 2 protein is much more highly expressed in ovaries than in total female flies. In males, Importin 2 protein is present at low levels in testes, at about the concentration found in whole adult females, but is undetectable in adult males (see below). In general, then, Importin 2 protein appears to be enriched in ovaries and testes relative to whole flies. However, the very high level of importin 2 mRNA in the testes (Fig 3B) is out of proportion to the relatively low level of Importin 2 protein (see also TOROK et al. 1995 ). These results suggest the possibility that translation of importin 2 mRNA is negatively regulated in the testes.

To further examine Importin 2 expression, the protein was localized by immunofluorescence in wild-type and 2^-/- testes. These data clearly show that in wild-type testes Importin 2 is highly expressed in immature germ cells located at the apical tip (Fig 4, A–C), where it is predominantly cytoplasmic (Fig 4, D–F), and is undetectable in more mature elongated sperm bundles (Fig 4, G–I). At the apical tip of testes premeitoic stem cells and spermatogonial cells exhibit stronger Hoechst fluorescence than postmeiotic spermatocytes (GONCZY et al. 1997 ). On the basis of this criterion, we conclude that Importin 2 is preferentially localized to stem and spermatogonial cells. The observed fluorescence is specific for Importin 2 since it is absent in 2^-/- testes (Fig 4, J–L). These results suggest that Importin 2 functions during the earliest stages of sperm development.

View larger version (57K): In this window In a new window Download PPT slide   Figure 4. Selective localization of Importin 2 in testes. Testes dissected from wild-type (A–I) or 2-/- (J–L) males were stained for DNA with Hoechst (A, D, G, and J) and Importin 2 (B, E, H, and K) with anti-Importin 2 antibodies (MATERIALS AND METHODS). Overlays are shown in C, F, I, and L. Confocal images were taken of the apical tip of wild-type (A–F) and 2-/- (J–L) testes and elongated sperm bundles (G–I) from wild-type testes. Bars, 100 µm.

Rescue of 2^-/- male sterility with importin transgenes:
The coexpression of importin 1, 2, and 3 in the testes raises the possibility that the occurrence of some fertile 2^-/- males could be due to the capacity of endogenous Importin 1 and/or 3 to partially perform the function(s) of the lost Importin 2. If true, then the ectopic expression of importin 1 and 3 transgenes in the testes might raise their levels enough to more efficiently rescue the 2^-/- sterility phenotype. Alternatively, if Importin 2 plays a paralog-specific role that is important but not essential in spermatogenesis, then importin 1 or 3 transgenes would not rescue male sterility. The capacity of each of the three importin genes to rescue male sterility was determined using the UASt transgene system (BRAND and PERRIMON 1993 ) with a Gal4^Hsp70 driver to ectopically express importin 1, 2, or 3 transgenes (MATERIALS AND METHODS). As shown in Table 2, 2^-/- males containing UASt importin 1, 2, or 3 transgenes and the Gal4^Hsp70 driver were significantly more fertile than sibling control males lacking either the transgenes or the driver. These results indicate that the activity(s) of importin 2 that is important for spermatogenesis can be replaced by elevating the expression levels of importin 1 or 3. Furthermore, testes squashes from 4- to 5-day-old flies demonstrated that each of the three transgenes partially rescued the sperm motility defect of 2^-/- males (data not shown). Specifically, testes from rescued flies contained significantly greater numbers of motile sperm than those from 2^-/- testes. In this experiment, flies received a single 2-hr heat-shock treatment. Repeated heat-shock treatments throughout development increased the rescue efficiency of the transgenes: 100% fertility (six of six males) for importin 1, 89% fertility (eight of nine males) for importin 2, and 78% fertility (seven of nine males) for importin 3. We conclude that the requirement for importin 2 in spermatogenesis is not due to a unique biochemical property of Importin 2 that is completely lacking from Importin 1 and 3.

Rescue of the 2^-/- female sterility with importin transgenes:
We next investigated the capacity of UASp importin 1, 2, and 3 transgenes to rescue the sterility defect in 2^-/- female flies. The Gal4^nanos-VP16 driver was used to express UASp importin transgenes in the female germline (RORTH 1998 ). The UASp importin 2 transgene efficiently rescued the 2^-/- sterility defect to the level found in 2^+/- females (Table 2). Eggs laid by importin 2-transgene-expressing 2^-/- females were morphologically wild type (not shown). Control sibling 2^-/- flies were sterile and laid defective eggs. As in males, the capacity of an importin 2 transgene to efficiently rescue the sterility of 2^-/- females is strong evidence that the phenotype is caused solely by a mutation in the importin 2 gene and not to a second, unlinked mutation. Interestingly, UASp importin 1 and 3 transgenes did not rescue the sterility of 2^-/- females (Table 2). Also, the characteristic deflated morphology of eggs laid by 2^-/- flies was unaffected by the ectopic expression of either importin 1 or 3 transgenes (data not shown).

To control for transgene expression levels, importin 1, 2, and 3 transgene mRNA levels were determined by Northern blot using a K10 3' UTR probe that is common to all UASp-expressed mRNAs (RORTH 1998 ). Ribosomal protein RP49 mRNA levels were used as loading controls. Importin transgene expression levels were quantified and normalized to RP49 levels by phosphoimaging. As shown in Fig 5A, the importin 1 transgene was expressed 2.3 times higher than the rescuing importin 2 transgene. Initially, we found that single importin 3 transgenes were expressed at levels slightly less than the importin 2 transgene. Therefore, flies containing two importin 3 transgenes were created. The combined expression of both transgenes raised importin 3 mRNA levels in the ovary to 1.4 times that of the importin 2 transgene. The 2^-/- females that expressed two copies of importin 3 were still completely sterile (0/20 fertility) and laid morphologically defective eggs that never hatched. These results argue that the failure of importin 1 and 3 transgenes to rescue the importin 2^D14 female sterility was not due to poor transcription of the transgenes, which, in both cases, exceeded that of a rescuing importin 2 transgene.

View larger version (58K): In this window In a new window Download PPT slide   Figure 5. Importin 1, 2, and 3 transgene expression. (A) A total of 15 µg of total RNA isolated from dissected ovaries of the indicated genotype (MATERIALS AND METHODS) was hybridized with a K10 3' UTR probe that is common to all UASp-derived transgenes and a RP49 probe as an internal loading control. Transgene expression levels were determined by normalizing the K10 3' UTR band to the RP49 band by phosphoimaging and are indicated relative to the expression level of the rescuing importin 2 transgene. (B) A total of 10 µg of protein isolated from dissected ovaries of the indicated genotype was examined by Western blot with anti-Drosophila Importin 2, 3, or anti--tubulin antibodies (MATERIALS AND METHODS). * indicates the slower migrating anti-Importin 3-crossreactive band that appears only when 1 is overexpressed. The Importin 2 S56A protein was made by changing the serine at position 56 of 2 to alanine.

The levels of importin proteins in dissected ovaries from transgenic flies were determined by Western blot using available anti-Importin 2 and anti-Importin 3 antisera. As shown in Fig 5B, ovarian Importin 2 migrates as a doublet that is absent from 2^-/- ovaries and is mostly replenished by expression of a UASp importin 2 transgene. In flies expressing two copies of the importin 3 transgene, the level of Importin 3 protein increased approximately five times over wild-type levels. The level of Importin 1 could not be directly determined since anti-Importin 1 antibodies are not currently available. However, because the untranslated sequences of the importin 1 and 2 transgenes differ by only 7 nucleotides (MATERIALS AND METHODS), it is unlikely that they are differentially translated. In addition, a slower migrating anti-Importin 3-crossreactive band always appeared in extracts from ovaries expressing a UASp importin 1 transgene (Fig 5B). Because Drosophila Importin 1 is 3300 daltons larger than Importin 3 it is possible that this band is Importin 1. Taken together, these results indicate that importin 1 and 3 transgenes are both highly expressed in the ovary. We conclude that Importin 2 has a unique role(s) in oogenesis that cannot be performed in vivo by either Importin 1 or 3.

Curiously, it appears that Importin 3 protein levels in the ovary varied in inverse proportion to Importin 2 protein levels. For example, Importin 3 levels were higher in the ovaries of 2^-/- flies than in wild-type flies (Fig 5B). Importin 3 levels decreased in 2^-/- ovaries in flies expressing an importin 2 transgene (Fig 5B), and the decrease was proportional to the level of transgene expression (not shown). Thus, it is possible that the expression of Importin 3 is influenced by the level of Importin 2.

The conserved Importin 2 phosphorylation site is not required for fertility:
Importin 2 isolated from ovaries and preblastoderm embryos is partially phosphorylated and migrates as a doublet on Western blots (Fig 3B and Fig 5B; KUSSEL and FRASCH 1995 ; TOROK et al. 1995 ; MATHE et al. 2000 ). Consistent with this, the upper band disappears upon phosphatase treatment (TOROK et al. 1995 ). The S. cerevisiae importin 1 homolog Srp1p is phosphorylated at serine-67 just downstream of the IBB domain. However, mutation of this site resulted in no discernable phenotypes (AZUMA et al. 1997 ). This serine is conserved, but not necessarily phosphorylated, in most conventional importin 's (AZUMA et al. 1997 ), including the three Drosophila importin 's. TOROK et al. 1995 speculated that Drosophila Importin 2 was phosphorylated at this site (serine-56) due to its consensus cdc2 phosphorylation sequence. We tested their hypothesis by introducing a mutant importin 2 transgene containing alanine instead of serine at position 56 (S56A). As shown in Fig 5B, ovarian Importin 2 S56A appeared as a single band that comigrated with the lower of the two wild-type Importin 2 bands. We conclude that serine-56 in Importin 2 is probably phosphorylated; however, we cannot rule out the possibility that serine-56 is required for the phosphorylation of some other residue. Interestingly, an importin 2 S56A transgene efficiently rescued 2^-/- female sterility. Specifically, 90% (18/20) of 2^-/- females expressing an importin 2 S56A transgene were fertile, compared with 0% (0/20) of 2^-/- females containing the transgene but not the Gal4^nanos-VP16 driver. In the same cross, 90% (18/20) of 2^+/- females expressing importin 2 S56A were fertile. These results demonstrate that, as in yeast, the phosphorylation of Importin 2 is not required for an essential in vivo function. Therefore, the paralog-specific role of Importin 2 in female gametogenesis is not likely due to its phosphorylation.

Effect of importin gene dosage on fertility:
The finding that all three importin transgenes rescued 2^-/- male sterility is consistent with the notion that a threshold concentration of some combination of the three importin 's normally perform an important function that any single paralog is capable of performing when ectopically expressed. If true, then the partial reduction in the concentration of any two importin 's might also cause sterility. This was tested by simultaneously reducing the gene dosage from two to one for two different importin genes. Because discrete mutations in either importin 1 or 3 genes were not available, we employed chromosomal deficiencies in combination with the importin 2^D14 allele. Males and females heterozygous for both importin 2^D14 and deficiencies that uncovered either the importin 1 (Df (3L) kto2) or the importin 3 (Df (3R) by416) genes (analysis not shown) were tested for fertility. Male flies heterozygous for importin 1 and 2 were significantly less fertile than males heterozygous for only the importin 2^D14 allele or the importin 1 deficiency alone (Table 1). Importin 1 and 2 double heterozygotes also produced greatly reduced numbers of motile sperm (not shown). In contrast, males heterozygous for importin 2 and 3 genes exhibited wild-type levels of fertility (Table 1). Females heterozygous for the importin 2^D14 mutation and the importin 1 or 3 deficiencies were fully fertile (Table 1) and laid morphologically wild-type eggs. Thus, spermatogenesis appears to be more sensitive than oogenesis to overall importin levels.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Animals contain both conserved (conventional) and organism-specific (nonconventional) importin genes (Fig 1). Little is known about the individual physiological roles of the various animal importin 's or their functional interplay in vivo. Conventional importin genes fall into three clades designated 1, 2, and 3 (Fig 1; see also MALIK et al. 1997 ; KOHLER et al. 1997 , KOHLER et al. 1999 ). Here we report the cloning and phylogenetic analysis of the first complete set of importin 1, 2, and 3 genes from an invertebrate, D. melanogaster, thereby demonstrating that the conventional importin gene family arose in animals prior to the split between invertebrates and vertebrates. On the basis of parsimony arguments and the fact that importin 2 and 3 genes are more similar to one another than to 1 genes (Fig 1), the phylogeny supports, but does not prove, that importin 2 and 3 genes arose from an 1 progenitor(s). Less likely is the alternative hypothesis that importin 2 and 3 progenitors were lost from fungal and plant lineages (ARAVIND et al. 2000 ). The C. elegans genome, which contains a single conventional importin 3 (IMA3) and two divergent worm-specific genes (GELES and ADAM 2001 ), shows that the conventional importin gene family is not strictly conserved in all animals. However, it is possible that the two nonconventional worm importin genes (IMA1 and -2) derive from progenitor 1 and 2 genes. Because IMA1 and IMA2 are restricted to the germline, they may have evolved as rapidly as have many sex determination genes (DE BONO and HODGKIN 1996 ; HANSEN and PILGRIM 1999 ).

The phylogeny of the conventional importin gene family (Fig 1) supports the adoption of a numbering scheme—importin 1, 2, and 3—to designate to which conserved clades a particular importin gene belongs. Additional levels of gene duplication and (presumably) functional specialization have in many species given rise to additional conventional isoforms. For example, Schizosaccharomyces pombe contains two importin 1 isoforms, and humans contain a total of eight importin 1, 2, and 3 genes. However, because it is not always clear how, or if, isoforms within individual clades are related among species, the current phylogenetic analysis does not support a numbering system for individual isoforms (e.g., importin 1-a, 1-b, 1-c, etc.).

The presence in the Drosophila genome of a single representative of each conventional clade simplifies the analysis of conventional animal importin genes and argues that flies are the most suitable genetic system for investigating their functional relationships in vivo. Previous genetic studies in Drosophila addressed the in vivo functions of importin 2 and 3 genes (KUSSEL and FRASCH 1995 ; TOROK et al. 1995 ; MATHE et al. 2000 ). The initial description of overgrowth of hematopoietic organs in homozygous importin 2 mutant flies (KUSSEL and FRASCH 1995 ; TOROK et al. 1995 ) proved to be incorrect (G. ADAM and I. KISS, personal communication). Similarly, we found that the mutant phenotypes ascribed to the insertion of a P element upstream of importin 3 (MATHE et al. 2000 ) could be recombined away from the P element (A. MASON, unpublished observations).

In our study, bona fide importin 2 null alleles provided by I. Kiss were used to investigate functional interactions among the conventional Drosophila importin genes during gametogenesis. Interestingly, the only phenotypes associated with homozygous null importin 2 mutations are male and female sterility. However, we cannot rule out the possibility that maternally derived Importin 2 also serves a role in early embryonic development, since oogenesis is disrupted in females lacking Importin 2. In gametogenesis, we have found that importin 1 and 3 transgenes can replace importin 2 during spermatogenesis but not during oogenesis. These rescue experiments suggest that Importin 2 has distinct roles during male and female gametogenesis.

Importin 2^-/- males exhibit defects in sperm individualization, produce very few motile sperm, and most flies are completely sterile. Sperm individualization appears to function as a quality