INTRODUCTION

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The nuclear lamina is a proteinaceous network underlying the nucleoplasmic side of the inner nuclear membrane (reviewed in references 9, 10, and 41). It is hypothesized to provide the structural framework for the nuclear envelope as well as anchoring sites for interphase chromosomes, thus organizing the structure of the nucleus and chromatin (reviewed in references 9, 10, and 41). The major components of the lamina are lamins, a family of intermediate filament-like proteins (see references 8, 35, and 38 for reviews of lamins). The lamina also contains nonlamin components. Examples are the Drosophila melanogaster Young Arrest (YA) protein (26, 30) and the vertebrate MAN antigens (44).

To better understand the molecular roles of nuclear lamina proteins, it is important to identify functionally important regions of these proteins. Several approaches have been taken to this question (11, 12, 17, 33, 54, 56, 60-62). However, these previous studies were carried out by necessity either in vitro or in yeast, which might not accurately reflect in vivo conditions in the metazoan lamina, or by ectopic expression in tissue culture cells, in which endogenous protein could affect the results seen with the ectopically expressed protein variants. Since the YA protein plays a developmentally essential role, and mutations that eliminate its function therefore arrest development, YA provides a unique example in which the functional domains of a nuclear lamina protein can be dissected in its normal in vivo environment and in the absence of any residual function of the protein.

YA is a maternally provided protein whose action is required at the start of Drosophila embryonic development (26, 29). Loss of YA function causes female sterility, with zygotes from homozygous mutant mothers arresting very early in development (26, 29). YA is present in developing female germ cells, postmeiotic oocytes, and 0- to 2-h (cleavage-stage) embryos (26, 28-30, 52). YA's localization in cleavage nuclei of embryos is cell cycle dependent: YA is in the nuclear lamina from interphase through metaphase but dissociates from the nuclear periphery at anaphase and telophase (26). Phenotypic analysis suggests that YA function is required for the transition from meiosis to mitosis and that YA may play a role in organizing chromatin structure and coordinating the nuclear activities required for the first mitotic division (29). Upon ectopic expression, YA associates with polytene chromosomes (32), further supporting a role of YA in organizing chromosomes within the nucleus. In transgenic flies, a wild-type Ya cDNA can fully rescue the null Ya² mutation (30). Therefore, using transgenic flies carrying different mutant forms of YA, classic complementation assays can be used to identify functionally important regions of YA. We have taken this approach to target potential functional domains of YA and to determine their roles in vivo.

The regions that we chose for targeted mutagenesis were largely based on examining the predicted YA sequence. Though YA protein shows no significant similarity to other known proteins in the database, it contains several motifs that might be of functional relevance (26, 29): YA has two putative nuclear localization signals, consistent with its being in the nuclear lamina. It also contains two potential Cys₂-His₂-type zinc fingers and an SPKK motif, which are known DNA binding motifs (1, 4, 5, 14, 55) and thus might mediate YA's function in organizing chromosomes within the nucleus by binding to DNA. YA also contains a glutamine (Q)-rich region, a serine/threonine (S/T)-rich region, and a highly positively charged C terminus, which are potential regions for protein-protein interaction (18, 43) and thus could mediate interactions between YA and other proteins in the nuclear envelope or in chromosomes. Finally, there are many potential phosphorylation sites in the YA protein, including the S/T-rich region and two sites (IT⁴⁴³PIR and FS⁵¹¹PKK) that match consensus for maturation-promoting factor (MPF) phosphorylation target sites (45); ITPIR also matches consensus for phosphorylation by mitogen-activated protein kinase (MAPK) (13). Since YA's nuclear envelope localization is cell cycle dependent, FSPKK and ITPIR could be potential targets for kinases that regulate the cell cycle-dependent activity of YA.

The results that we report here on transgenic flies carrying deletion and substitution mutations in various regions of YA identify several regions important for YA function in vivo, including YA's N and C termini, the cysteine residues in the two potential zinc fingers, and one potential MPF and MAPK phosphorylation site (ITPIR). We also developed an assay for biochemically probing YA-YA interactions in vivo and showed that YA proteins can interact with one another. These results confirmed and extended our previous (29) and present findings of genetic interactions between different Ya alleles. Finally, we showed that the C-terminal 179 amino acids of YA are needed for targeting YA to, or retaining YA in, the nuclear envelope.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Construction of epitope-tagged and targeted mutant Ya constructs. All epitope-tagged and targeted mutant constructs were made by using the wild-type Ya cDNA described by Lopez et al. (30). This cDNA was cloned into either pUC19 or M13 vectors for further mutagenesis. A combination of standard site-directed mutagenesis, PCR, and cloning techniques was used to generate the epitope-tagged or targeted deletion or substitution constructs. Details of how each mutant was generated, and primer sequences, are described in reference 27 and are available upon request. The constructs are summarized in Fig. 1. All tagged constructs and targeted mutant Ya constructs were verified by sequencing both before and after being cloned into the EcoRI site of the P-element vector pW5g26 (30) for fly germ line transformation. This cloning places the mutated Ya cDNAs under the control of the Drosophila hsp26 promoter.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Schematic diagrams and summary of complementation results for all mutant constructs. YA proteins encoded by each mutant construct discussed in this report are shown schematically under a diagram of wild-type YA with motifs indicated. Numbers of independent lines obtained for each mutant construct are also shown, and all lines were analyzed. The position of a mutation is indicated by an asterisk; the letter above the asterisk is the amino acid change introduced. V, a delection; A... .A, two alanine residues with several amino acids in between. Results of complementation of the apparent null mutation Ya2 or the leaky alleles Ya77 (an N-terminal lesion [29]) and Ya70 (a C-terminal lesion [29]) by each mutant protein are also summarized. Complementation was tested either without heat shock (NHS) or with heat shock (HS) as described in Materials and Methods. Levels of complementation were scaled from  to +++++. , no complementation (no progeny were produced); +, very weak rescue (only pupae or at most one or two adult progeny were produced); +++++, complete rescue, as indicated both by production of progeny numbers comparable to that of sense Ya cDNA as well as by completely wild-type-like embryonic phenotype of embryos from rescued females. ++, +++, and ++++ represent intermediate amounts of complementation (approximately 10 to 30%, 30 to 60%, and 60 to 80%, respectively, of the level for complete rescue). Q4.28.1.1 is a mutation which results in a truncation of the C-terminal 179 amino acids of the YA protein in addition to the Q-rich region deletion.

Fly germ line transformation. The N(HA)1, N(HA)3, C(HA)3, ITPIR, FSPKK, and ITPIR-FSPKK constructs were injected into w; 2-3 Sb/TM6 embryos (48). All other constructs were injected into w; 2-3(99B) embryos, using the protocol of Park and Lim (42) with small modifications. G₀ flies were crossed to z¹w^11E4 flies for several generations to generate stable transformant lines carrying each construct. Independent lines for each construct were verified by genomic Southern blotting, and at least two independent single-insert lines per construct were tested unless otherwise noted (Fig. 1).

Complementation assays. Male flies carrying each mutant construct were crossed to one of the following three kinds of virgin females: XX Ya² [XX, y² Ya² w^bf spl sn³/Y, y⁺ Ya⁺ w⁺ B^s], XX Ya⁷⁷ [XX, y² Ya⁷⁷ cv v f/Y, y⁺ Ya⁺ w⁺ B^s], and XX Ya⁷⁰ [XX, y² Ya⁷⁰ cv/Y, y⁺ Ya⁺ w⁺ B^s]. Ya² is an apparent null allele (26), whereas Ya⁷⁰ and Ya⁷⁷ are two leaky but complementing Ya alleles (29, 37). Ten female progeny from each of these crosses were checked for fertility as specified by Lopez et al. (30), but either without heat shock or with heat shock. For heat shock treatment, the progeny from the above crosses were heat shocked either three times a day for 3 days or once a day for 3 days, each time at 37°C for 1 h. For any fertile crosses, the resulting progeny were examined to verify that they were of the right phenotype. Male flies carrying the sense or antisense Ya cDNA construct (30) were used as either positive or negative controls, respectively. Since flies carrying the N(HA)1 construct are homozygous, and behave exactly like flies carrying the sense Ya cDNA construct based on both female fertility and embryonic phenotypes (see Results), these flies were also used as wild-type controls for some of the experiments described below. For each mutant construct, all independent lines were tested a minimum of three times in each of the three complementation assays. Ovaries from the females used for complementation of Ya² were also dissected and subjected to Western blot analysis in order to correlate levels of transgene expression with levels of complementation.

Immunofluorescence. Embryos from females carrying a given mutant construct in the Ya² background were collected over a 0- to 2-h period, fixed, and stained with 4',6-diamidino-2-phenylindole (DAPI) and anti-YA antibodies as described by Lopez et al. (30).

Immunoprecipitation and Western blot analysis. Transgenic flies carrying one copy of either the N(HA)3 or the C(HA)3 construct (orange-colored eyes) were crossed to transgenic flies carrying one copy of the sense Ya cDNA or each mutant Ya construct (also orange-colored eyes). Red-eyed male flies, which contained both the hemagglutinin (HA)-tagged construct and the sense Ya cDNA or the mutant construct, were used as sources of proteins for immunoprecipitation assays. These red-eyed male flies were heat shocked at 37°C for 1 h and allowed to recover at room temperature for 1 h. Then they were homogenized in 1× lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, 20 mM Tris-HCl [pH 7.5], 50 mM NaCl), using 10 µl of buffer per fly. The homogenates were centrifuged at 4°C for 5 min. The supernatant was then incubated on ice for 1 to 2 h with either anti-YA antibodies or monoclonal anti-HA antibody (Boehringer Mannheim) followed by incubation with Pansorbin beads (Calbiochem) for 1 to 2 h on ice. The beads were spun down, washed eight times, each time with 1 ml of lysis buffer, boiled in sodium dodecyl sulfate (SDS) sample buffer (26), and loaded on an SDS-7.5% polyacrylamide gel (6 fly-equivalents per lane). Proteins were transferred to nitrocellulose filters and probed with affinity-purified anti-YA antibodies (26). For detection, the enhanced chemiluminescence Western blotting system (Amersham Corp.) was used as described in the supplier's instruction manual.

Assays for position effect variegation (PEV) and viability. Homozygous males (except in the case of the C1-C2 and C1-C2-FAPKK mutations, when heterozygous males were used) carrying each mutant construct were crossed to In(1)l^v231/FM6 virgin females (6). Eggs produced in a 48-h period were heat shocked once a day at 37°C for 1 h, for the subsequent 10 days of fly development. Progeny were counted. The absolute male viability (AMV) was calculated as numbers of In(1)l^v231/Y males over the number of total females. The relative male viability was calculated as AMV for each construct over AMV for either the antisense Ya cDNA construct or the N(HA)1 construct. Calculations were done according to Dimitri and Pisano (6).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

We made all of the targeted mutations in the wild-type, untagged Ya cDNA. These constructs are summarized in Fig. 1. They include mutations in potential regions for DNA binding (C1, C2, C1-C2, FSPKK, FAPKK, C2-FAPKK, and C1-C2-FAPKK), protein-protein interaction (Q, S/T, and Q-S/T) and phosphorylation (ITPIR, FSPKK, ITPIR-FSPKK, IAPIR, FAPKK, IAPIR-FAPKK, FEPKK, S/T, and SY160). We used the untagged Ya cDNA to make the mutant constructs because all the terminally HA-tagged Ya constructs which we initially made for functional dissection analysis could not be used for our purposes. Those initial tagged constructs contained one or three copies of the influenza virus nine-amino-acid HA tag (22, 59) at either end of the YA protein [N(HA)1, N(HA)3 and C(HA)3]. YA proteins produced by N(HA)3 or C(HA)3 were nonfunctional, and the YA protein produced by the N(HA)1 construct, although fully capable of rescue of Ya mutations, was not detectable by anti-HA antibodies.

All of the epitope-tagged and targeted mutant constructs were introduced into flies through germ line transformation (as described in Materials and Methods), and stable transformant lines were obtained for all constructs (Fig. 1). In all cases but one, there were no effects of the constructs on viability and fertility of Ya⁺ flies carrying the transgene, in agreement with results reported for the wild-type Ya cDNA (sense Ya cDNA [30]). The one exception was that mutations in the ITPIR motif caused dominant lethality upon ectopic expression in early z¹w^11E4 pupae; however, in other genetic backgrounds, ectopic expression of this mutant YA had no effect on viability or fertility of the transgenic animals (27). To further test for effects of ectopic expression of all the constructs shown in Fig. 1, we implemented the assay of PEV, which is very sensitive to the organization of chromosomes (reviewed in references 53 and 57). Lopez (31) had shown previously that ectopic expression of wild-type YA has no effect on the level of male lethality associated with the variegating lethal In(1)l^v231 (6). We tested whether the lethality of In(1)l^v231 was suppressed or enhanced by the ectopic addition of the mutant YA proteins. We observed that ectopic expression of any mutant YA proteins similarly had no effect on PEV (data not shown). We then went on to characterize the function of all the mutant YA proteins.

Targeted mutations identified regions of the YA protein important for its function. The in vivo functions of all constructs were tested by their ability to rescue progeny production by the putative null Ya² mutation. Except for N(HA)1, YA proteins from all mutant constructs were present at levels comparable to those from control flies carrying the sense Ya cDNA construct (examples are shown in Fig. 2). Complementation tests were carried out under conditions either without heat shock or with heat shock (as described in Materials and Methods). Heat shock treatment increased the level of expression of the Ya transgene in ovaries above the non-heat shock basal level driven by the hsp26 promoter in the constructs (data not shown). For some partially functional mutants, the higher level of expression improves their ability to complement Ya mutations (Fig. 1). Flies carrying the fully functional sense Ya cDNA construct (30) were used as positive controls. Results from these analyses, summarized in Fig. 1, have allowed the identification of regions important for YA function, as described below.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   Western blots of male transgenic flies carrying certain targeted mutations. Extracts from heat-shocked transgenic male flies were subjected to electrophoresis on an SDS-7.5% polyacrylamide gel. Bradford assays were used to ensure that the same amount of total protein (75 µg) was loaded in each lane. A Western blot of the gel was probed with anti-YA antibodies as described in Materials and Methods. Proteins from flies carrying the antisense Ya cDNA, the sense Ya cDNA, Q, Q4.28.1.1, S/T, and Q-S/T (two different lines) were probed. While full-length YA from transgenic males has an apparent molecular mass of 97 kDa, and Q, S/T, and Q-S/T have apparent molecular masses of 96, 87, and 86 kDa, as predicted, Q4.28.1.1 produces a YA protein of only 70 kDa.

N-terminal mutations. N(HA)1, which has only nine amino acids of the HA tag inserted right after the ATG, fully complemented Ya². However, when 48 amino acids (three copies of the HA tag and associated linker sequences) were inserted at the same position, in N(HA)3, the mutant protein could rescue Ya² only very weakly. This finding suggests that the N terminus of YA protein is very sensitive to perturbations.

Q-rich and S/T-rich regions. Q-rich and S/T-rich regions are potential regions for protein-protein interaction (18, 43). Their importance in YA function was thus tested by deletions of each region. Q, which has a deletion of 12 amino acids of the Q-rich region, fully complemented Ya², suggesting that the Q-rich region is not essential for YA function. In contrast, S/T, which has a deletion of 99 amino acids of the S/T-rich region, can complement Ya² only very weakly. Therefore, the S/T-rich region is required for YA function. Deletions of both the Q-rich region and the S/T-rich region (Q-S/T) behaved similarly to the S/T mutation by itself, giving very weak complementation of Ya², consistent with the conclusion that the Q-rich region is not required for YA function.

Potential MPF phosphorylation sites (ITPIR and FSPKK). Two sets of mutants were made to test the importance of the two motifs that match the consensus of MPF phosphorylation sites in YA. We deleted each or both of the motifs (ITPIR, FSPKK, and ITPIR-FSPKK) and made alanine substitution mutations at the serine or threonine residue (IAPIR, FAPKK, and IAPIR-FAPKK) to further test their roles in phosphorylation. Since FSPKK is part of YA's potential nuclear localization signal, we also made a glutamic acid substitution of the serine residue in the FSPKK motif (FEPKK) to test its involvement in regulating nuclear entry of YA. Dephosphorylation of the Xenopus transcription factor NF7 (nuclear factor 7) in its nuclear localization signal allows the protein's entry to the nucleus, whereas changing the phosphorylation site to a glutamic acid to mimic the negative charge of phosphorylation blocks entry of the protein to the nucleus (26).

C-terminal mutations. The C terminus of the YA protein also is sensitive to perturbations and is important for YA function: C(HA)3, which has three copies of the nine-amino-acid HA tag (plus linker sequences) inserted immediately before the stop codon, almost completely abolishes YA function (Fig. 1).

Targeted mutations defined separable domains of the YA protein. Although complementation of Ya² is the most stringent test for function of a mutant protein, the existence of interallelic complementation of Ya alleles (29, 37) permits tests for whether portions of the protein have functionality even if the protein as a whole is nonfunctional. Therefore, we tested each mutant protein for its ability to complement the Ya⁷⁷ and Ya⁷⁰ mutations, which are leaky mutations at the N and C termini, respectively, of YA (29). As for complementation of Ya², we also used both non-heat shock and heat shock conditions. The results are summarized in Fig. 1. As expected, all mutant proteins that can fully complement Ya² also complemented Ya⁷⁰ and Ya⁷⁷ (Fig. 1). These include N(HA)1, Q, SY160, FSPKK, FAPKK, and FEPKK. Therefore, we will concentrate only on the other mutants here.

N-terminal mutations. All of the N-terminal mutations either complemented very weakly [N(HA)3] or failed to complement (C1 and C2) the N-terminal mutation Ya⁷⁷. However, they all complemented the C-terminal mutation Ya⁷⁰ significantly better. For example, C1, which did not complement Ya⁷⁷, fully complemented Ya⁷⁰. Similar results were obtained for C2, though a lower level of complementation of Ya⁷⁰ was seen. This result suggests that mutation of C2 has a more dramatic effect on YA function than that of C1. The double mutation C2-SY160 or C1-C2 behaved like the C2 mutation alone, suggesting that SY160 is not important for YA function; furthermore, simultaneous mutations of the two potential zinc fingers did not have a detectable synergistic effect. The fact that these N-terminal mutations complement the C-terminal Ya⁷⁰ mutation indicates that these mutations retained a functional C-terminal domain.

S/T-rich regions. Both S/T and Q-S/T only weakly complemented Ya⁷⁰, but they fully complemented the Ya⁷⁷ mutation. Therefore, these mutant proteins might have a more defective C-terminal domain. This suggests that the C-terminal domain of YA extends from amino acid 336 (beginning of the S/T-rich deletion) to the C-terminal end.

Mutations at the ITPIR motif. Both ITPIR and IAPIR mutations complemented Ya⁷⁰ and Ya⁷⁷, in contrast to their weak or absent complementation of Ya². In addition, the ITPIR-FSPKK or IAPIR-FAPKK mutation behaved similarly to the ITPIR or IAPIR mutation, respectively, in this assay. These results suggest that mutations in the ITPIR motif still retain function of both the N and C domains.

C-terminal mutations. C(HA)3 complemented Ya⁷⁰ and Ya² poorly but complemented Ya⁷⁷ much better. This is the reverse of the situation with N(HA)3, which rescued Ya⁷⁰ better than Ya⁷⁷. These results indicate that C(HA)3 still retains partial N-terminal function.

Heterozygous combinations of N(HA)3 and C(HA)3 complemented Ya². Since all of the N-terminal mutations complemented the C-terminal mutations, we also tested whether N(HA)3 and C(HA)3 in heterozygous combination was functional by testing whether this combination could rescue the Ya² mutation. As shown in Fig. 1, neither tagged protein by itself could complement the Ya² mutation without heat shock. However, flies carrying both N(HA)3 and C(HA)3, even without heat shock treatment, could rescue the sterility of Ya² (data not shown).

YA proteins coimmunoprecipitate. One model to explain the above intragenic complementation is that YA proteins form complexes with each other and that the combination of a functional N terminus and a functional C terminus in trans can allow YA function. To test this, we developed a biochemical assay to directly determine whether YA associates with itself in vivo.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Immunoprecipitation of untagged YA or mutant YA associated with N(HA)3 or C(HA)3. (A) YA and N(HA)3. Transgenic male flies carrying either the sense Ya cDNA or N(HA)3 or both were extracted, and their proteins were subjected to immunoprecipitation. Western blots were probed with anti-YA antibodies as described in Materials and Methods. Lanes 1, 4, and 7 show YA proteins in extracts without immunoprecipitation. Lanes 3, 6, and 9 show proteins in immunoprecipitates with anti-HA antibody (+anti-HA). Note that untagged YA does not precipitate with this antibody (lane 3) except in the presence of N(HA)3 (lane 6). N(HA)3 is precipitated by anti-HA antibody in the presence or absence of untagged YA (lanes 6 and 9). Lanes 2, 5, and 8 show immunoprecipitation of YA (untagged or tagged) from these extracts to demonstrate that untagged YA is immunoprecipitable with anti-YA antibodies (+anti-YA). All lanes have the same amount of starting material. Results with YA and C(HA)3 are essentially the same (data not shown). (B) IAPIR-YA and N(HA)3. Transgenic male flies carrying one or both of IAPIR-YA and N(HA)3 were used for immunoprecipitations as described above. Results with IAPIR-YA and C(HA)3 are essentially the same (data not shown).

The C-terminal 179 amino acids are essential for the proper nuclear envelope localization of YA. YA function appears to correlate with its nuclear envelope localization (29). Therefore, failure of a mutant protein to be functional could be a result of its not being able to be properly localized to the nuclear envelope. To address this question, we tested all mutant proteins for the ability to localize to the nuclear envelope.

View larger version (92K): [in this window] [in a new window]   FIG. 4.   Localization of mutant YA proteins in embryos. Zero- to 2-h embryos were collected from females carrying the sense Ya cDNA (A to F) or FAPKK (G to L) construct in the Ya2 background and stained with DAPI (A, C, E, G, I, and K) and anti-YA antibodies (B, D, F, H, J, and L). FAPKK has a cell cycle-dependent nuclear envelope localization like that of the YA produced by the Ya sense cDNA construct. The same staining pattern was seen for all other mutant proteins that were tested in this assay. The bar represents 10 µm and applies to all panels.

View larger version (55K): [in this window] [in a new window]   FIG. 5.   Localization of mutant YA proteins in nuclei of male accessory glands and larval brain squashes. (A) Male accessory glands from transgenic flies carrying the sense Ya cDNA (A and B) or C2-FAPKK (C and D) construct were stained with DAPI (A and C) and anti-YA antibodies (B and D). The bar represents 10 µm and applies to all panels. (B) Larval brains from transgenic flies carrying the sense Ya cDNA (A to F) or C2-FAPKK (G to L) construct were stained with DAPI (A, C, E, G, I, and K) and anti-YA antibodies (B, D, F, H, J, and L). The bar represents 10 µm and applies to all panels. YA protein is visible in nuclear envelopes of accessory gland nuclei, as well as interphase and prophase nuclei in larval brain squashes. Staining of anaphase nuclei also shows weak nuclear envelope staining, but it appears more diffuse. The staining pattern shown here is the same as for flies carrying the sense Ya cDNA (30). A similar staining pattern was seen for all the mutant constructs listed in Fig. 1 except Q4.28.1.1. (C) Accessory glands (A and B) and larval brains (C and D) were dissected from Q4.28.1.1 heat-shocked males or larvae and stained with DAPI (A and C) and anti-YA antibodies (B and D). Stainings of brain squashes of larvae carrying the antisense Ya cDNA (E and F) are shown as a negative control. Photographs for panels C to F were taken using the same exposure time. Notice the nucleoplasm staining of YA in both tissues of Q4.28.1.1 and that the staining is excluded from the nucleolus. The bar represents 10 µm and applies to all panels.

All of the mutant proteins retain their ability to bind polytene chromosomes upon ectopic expression. Phenotypic analyses of Ya mutant eggs and embryos suggest that YA may be needed to organize chromosomes within the nucleus (29). YA protein is capable of associating with polytene chromosomes upon ectopic expression (using transgenic flies carrying the sense Ya cDNA [30]). Therefore, the loss of function of some mutant proteins could be due to their failure to interact with chromatin. We tested all of the nonfunctional mutant proteins for the ability to bind polytene chromosomes upon ectopic expression. As shown in Fig. 6, all mutant proteins, including those with single, double, or triple mutations of the potential DNA binding motifs, associate with polytene chromosomes. There are no obvious differences between the intensity or the banding pattern of staining of the control and any of the mutant proteins. Therefore, none of the mutant proteins, including those altering the potential DNA binding regions, is essential for polytene chromosome binding.

View larger version (122K): [in this window] [in a new window]   FIG. 6.   Localization of mutant YA proteins to polytene chromosomes of larval salivary glands. Polytene chromosomes from salivary glands of transgenic larvae carrying the sense Ya cDNA (A and B, as controls) or the transgene in Q4.28.1.1 line (C and D), as an example, were stained with DAPI (A and C) and anti-YA antibodies (B and D). The staining pattern seen for the sense Ya cDNA is as reported by Lopez and Wolfner (32); the staining pattern of Q4.28.1.1 is the same. For all mutant constructs listed in Fig. 1, a staining pattern similar to the one shown was observed. The bar denotes 20 µm and applies to all panels.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We used a genetic assay to define important regions of the Drosophila YA nuclear envelope protein in its normal in vivo context. Our studies were made possible by the existence of nonfunctional and partially functional alleles of ya, and by the absolute requirement for YA function to allow embryos to pass through a critical developmental transition. By introducing epitope-tagged and targeted Ya mutant constructs into flies and testing their abilities to complement the maternal lethality of existing Ya mutations, we identified regions important for YA function in vivo. To our knowledge, this is the first study of this type of a metazoan nuclear envelope protein. As mutations are identified in other metazoan nuclear envelope proteins (e.g., a partially functional Drosophila lamin Dm₀ mutant was reported recently [24]), this type of analysis can be extended to other nuclear envelope components.

Our results showed YA's function requires both its N- and C-terminal ends, the cysteine residues in its two potential zinc fingers, its S/T-rich region, and an ITPIR motif. Critical amino acids are distributed along the whole length of the YA molecule. Previous genetic analysis showed that an N-terminal Ya mutation can complement a C-terminal Ya mutation (29, 37). Here we used a set of mutations at either end of the YA protein to show that the complementation reported in the previous studies is not simply aberrant properties of two alleles; rather, YA has separable domains. In addition to noting the N- and C-terminal domains and distribution of essential elements in YA, we can draw several conclusions, as discussed below.

YA is present in complexes with other YA molecules. Ya mutant alleles show intragenic complementation, with all lesions at the N terminus complementing all alleles with C-terminal lesions (references 29 and 37 and this study). These results suggest that individual YA proteins have separable domains and that YA protein might interact with itself. Here, we provided biochemical evidence, in the form of coimmunoprecipitation, in support of this interaction in vivo (Fig. 3). Thus, both genetic and biochemical evidence suggest that YA proteins are in complexes with each other. At this point, we cannot distinguish if this interaction is direct or indirect.

The C terminus of YA regulates YA's nuclear envelope localization. The mutant YA protein in Q4.28.1.1 flies fails to be localized to the nuclear envelope, and this protein is nonfunctional in complementation rescue assays. These findings suggest that nuclear envelope localization is essential for YA function, consistent with previous studies showing that YA function is correlated with its nuclear envelope localization (29). The Q4.28.1.1 mutation causes a C-terminal 179-amino-acid truncation of the YA protein. This protein still contains one of the potential nuclear localization signals in the YA protein, and it is capable of entering the nucleus (Fig. 5). Therefore, the 179 amino acids missing in Q4.28.1.1 are essential for targeting YA to, or retaining YA in, the nuclear envelope.

ITPIR motif and phosphorylation. Two regions of the YA protein, ITPIR and FSPKK, match the consensus for MPF phosphorylation target sites (26, 45); ITPIR also matches consensus for a MAPK target site (13). Our mutational analysis showed that the ITPIR motif is required for YA function, though the FSPKK motif by itself is not. Deletions of the ITPIR motif, ITPIR and ITPIR-FSPKK, as well as substitution mutations of the threonine residues in this motif, IAPIR and IAPIR-FAPKK, all adversely affected the function of YA. In addition, mutations of the ITPIR motif cause dominant lethal effects upon ectopic expression during early pupal development in some genetic backgrounds (27). These results suggest that the threonine residue of ITPIR is important for YA function. Given that this site matches consensus for important developmental or cell cycle-regulated kinases, the threonine of ITPIR may be a site for regulated phosphorylation needed for YA's function or one of multiple phosphorylation sites that regulate YA's nuclear entry as in lamin (15, 45), Xenopus NF7 (25, 36, 50), and yeast SWI5 proteins (39, 40). Consistent with this suggestion, we have recently observed that YA is a phosphoprotein and that IAPIR-YA protein is less phosphorylated than wild-type YA (60a).

DNA binding and chromatin association. Phenotypic analysis of Ya mutant eggs and embryos suggests a role of YA in organizing chromatin structure (29). Consistent with this, ectopically expressed YA associates with polytene chromosomes (32). Since YA protein contains two Cys₂-His₂-type zinc fingers and an SPKK motif, which are potential DNA binding motifs (1, 4, 5, 14, 55), we initially expected that YA functioned by binding to DNA directly through these regions. Indeed, when the cysteine residues were substituted by alanines in either or both the potential zinc fingers, the protein lost its function. This result indicates that these cysteine residues are essential for YA function.