The Zinc Finger Protein Zfr1p Is Localized Specifically to Conjugation Junction and Required for Sexual Development in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.


Introduction
Cell junctions provide contact between neighboring cells or between a cell and the extracellular matrix in multicellular organisms. Intercellular junctions serve to maintain cell and tissue polarity and integrity, integrate intra-and intercellular signaling. In vertebrates, there are three major types of cell junctions, namely, anchoring, occluding, and communicating junctions. Anchoring junctions mechanically attach cells to their neighbors or to the extracellular matrix and organize the cortical cytoskeleton beneath the plasma membrane to modulate cell and tissue behavior [1]. Occluding junctions seal cells together in the epithelium in a way that prevents even small molecules from passing from one side of the sheet to the other [2,3]. Communicating junctions are aqueous intercellular channels that allow the diffusion of small molecules and ions from cell to cell [4]. The molecules responsible for creating cell junctions include various cell adhesion molecules, such as selectins, cadherins, integrins, and members of the immunoglobulin superfamily [5]. These core junctional components are also assisted by additional cell-type specific and accessory molecules, which cooperate to tailor junctions structurally and functionally [1].
In unicelluar protists, intercellular conjugation junctions are required for sexual reproduction. Conjugation is a highly conserved developmental process in ciliates that has been well studied [6,7]. In preparation for conjugation, Tetrahymena cells actively modify their pattern of protein synthesis. The anterior ends of the cells transform from a pointed to a blunt shape and from ciliated and ridged to smooth in texture [8]. During the course of conjugation, the conjugation junction undergoes dramatic membranous metamorphosis [8,9]. A number of cisternae are present between the junction and kinetosomes of the adoral zone of the membranelles [10,11]. In Tetrahymena, a cellcell junction is robust in order to survive the mechanical stresses experienced when two individual and highly mobile cells attempt to form a union [12]. An elaborate conjugation junction structure is also required for pronuclear exchange during the conjugation stage [13]. The pronuclear exchange is impelled by microtubulerich baskets whose terminals are connected with the junction. After a reciprocal nuclear exchange, the junction reestablishes integrity [10]. Using a proteomics-based approach, 15 proteins in the Tetrahymena conjugation junction structure have been identified. They include fenetrin, several cytoskeletal, nuclear, mitochondrial, ribosomal proteins and hypothetical proteins [12]. Fenetrin is a structural protein, it could facilitate the events surrounding the exchange of genetic material at the mating junction, by providing a structural scaffold at the junction between mating cells [12]. Recently, it has been reported that Cda13p associated with membrane trafficking is transiently localized on the resealed conjugation junction and participates in events associated with remodeling of the nuclear exchange junction during conjugation [14]. Although some conjugation junction structural proteins have been identified in Tetrahymena, the molecular mechanism underlying the process remains poorly understood.
Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, protein folding and assembly, protein-protein interactions as well as membrane association [15]. In higher eukaryotes, the zinc finger protein ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus. ZFPL1 interacts with the cis-Golgi matrix protein GM130 and is important for the integrity of cis-Golgi [16]. During C. elegans spermatogenesis, the zinc finger containing protein spe-10 is required for membranous organelles to properly deliver lysosome-related fibrous bodies to the C. elegans spermatid and its DHHC-CRD zinc finger motif is essential for this function [17]. Based on the published macronucleus genome and the gene expression profiles of T. thermophila [18,19], more than two hundred zinc finger domain containing genes have been predicted (http://www.ciliate.org; http://tfgd. ihb.ac.cn). One such zinc finger domain containing gene named ZFR1 (zinc finger-related protein, TTHERM_01285910) was identified in the present study. Immunofluorescence staining for Zfr1p showed that it was specifically localized in the conjugation junction structure. Deletion of its zinc finger and hydrophobic domains altered its localization. Truncated Zfr1p decorated foci located adjacent to the basal body. This localization pattern is consistent with the location of the Golgi apparatus, which is localized predominantly in the cell cortex and is closely associated with the mitochondria [20]. The mating pair of a ZFR1 knockout cell was less stable than that of a wild-type and ZFR1 over-expressing cell. Conjugation development of a somatic ZFR1 knockout cell was aborted 8-10 h into the mating stage, possibly due to defects in pronuclear exchange or reestablishment of conjugation junctions. These results showed that Zfr1p is required for the stability and integrity of conjugation junctions in Tetrahymena. Furthermore, Zfr1p is essential for the sexual life cycle of Tetrahymena.

Materials and Methods
Tetrahymena strains and culture conditions The wild-type B2086 (mating type II) and CU428 (micronuclear genotype Mpr/Mpr; mating type VII) strains of T. thermophila were provided by Dr. Peter. J. Bruns (Cornell University,Ithaca, NY, now available through the National Tetrahymena Stock Center, http://tetrahymena.vet.cornell.edu/index.html). The cells were grown in SPP medium [21] at 30uC. For analysis of conjugation, log-phase growing cells of different mating types were washed, starved (16-24 h at 30uC), and mixed in 10 mM Tris-HCl (pH 7.4) at equal amounts (,2610 5 cells/ml), as previously described [22].

Cloning of the ZFR1 gene
The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. EZL1, which is expressed specifically during conjugation stage, catalyzes scnRNA-dependent K27 methylation and is required for internal eliminated sequences elimination [23]. A total of 51 genes were found to be coexpressed with EZL1 [19]. One of the genes, TTHERM_01285910, which we chose to study and named ZFR1, was found to be co-expressed with EZL1 with a correlation coefficient of 0.997. The ZFR1 gene was first identified from the T. thermophila database (http://www. ciliate.org). The expression profile of this gene was also obtained from the T. thermophila microarray database (http://tfgd.ihb.ac.cn) [19]. Total RNA was extracted from Tetrahymena cells using Trizol (Takara) and was treated with RNase-free DNase I (Takara). The first-strand cDNA was synthesized using a PrimeScript TM reverse transcriptase (RT) and random hexamer primers. The ZFR1 cDNA was cloned and sequenced. Expression profile of ZFR1 was then confirmed by quantitative RT polymerase chain reaction (qRT-PCR). qRT-PCR was performed with the SYBR Premix Ex Taq TM (Takara) on a ABI StepOne Plus system (Applied Biosystems, USA). Each reaction was performed in triplicate. The values were normalized to the expression of the ribosomal 17S rRNA as an internal control. The primers qRT-Pup and qRT-Pdown were used (Table S1). The following parameters were used for PCR: 10 min at 95uC, followed by 40 cycles of 95uC for 15 s, 53uC for 30 s, and 68uC for 35 s. A melting curve of the PCR products (60-90uC) was also obtained to ensure the absence of artifacts.

Construction of somatic knockout ZFR1 strains
To create the targeting construct, the 59 and 39 flanking regions of ZFR1 were amplified from genomic DNA using the PCR primers KO-59 FW and KO-59 RV, and KO-39 FW and KO-39 RV (Table S1), respectively. The neo4 cassette conferring paromomycin sulfate resistance was amplified using primers neo4FW and neo4RV (Table S1). The ZFR1 knockout construct was obtained by overlapping PCR using primers KO-FW and KO-RV (Table S1). B2086 and CU428 cells were transformed with the respective constructs using the Biolistic PDS-1000/He particledelivery system (Bio-Rad), as previously described [24]. Transformants were selected on the basis of resistance to paromomycin. To obtain somatic ZFR1 knockout strains, the cells were subjected to stepwise selection in increasing concentrations of paromomycin sulfate in the presence of 0.1 mg/ml CdCl 2 , starting from 60 mg/ ml to a final concentration of 50 mg/ml until the cells failed to grow. The endogenous macronuclear ZFR1 gene was completely replaced by phenotypic assortment and selection in increasing concentrations of paromomycin sulfate. (The genotypes and phenotypes of all the strains used in this study are provided in Table S2).

Viability test
After 5-6 h of mating, 300 individual ZFR1 knockout cells and WT mating pairs were isolated and incubated in single SPP medium drops, as previously described [25,26]. Three hours later, the drops were examined to exclude cells that had been killed during pair isolation. After 48 h, the drops were re-examined. To check for complete conjugation in wild-type (B20866CU428) and ZFR1 knockout cells (gZFR1-B26 gZFR1-C4), the cells were incubated using 15 mg/ml 6-methylpurine (6-mp; Sigma) or 60 mg/ml paromomycin sulfate in SPP.

Construction of HA-ZFR1 strains
To create the hemagglutinin (HA)-ZFR1 construct, the HA coding sequence was inserted next to the initiation codon of ZFR1 by overlapping PCR. The primers used for the PCR were HA-59FW and HA-59RV, HA-39FW and HA-39RV, and HA-neo4FW and HA-NextRV (Table S1). The Neo4 cassette [27] was introduced into the 59 flanking sequence of HA-ZFR1 by PCR. The primers used for PCR were HA-neo4FW and HA-neo4RV, and neo4-59FW and neo4-39RV. The HA-ZFR1-neo4 construct was obtained by overlapping PCR with the primers HA-FW and HA-RV. CU428 and B2086 cells were transformed with the respective constructs using the Biolistic PDS-1000/He particle-delivery system (Bio-Rad Laboratories, USA), as previously described [24]. The endogenous macronuclear ZFR1 gene was completely replaced by phenotypic assortment and selection in increasing concentrations of paromomycin sulfate.

Western blot analysis
Whole-cell proteins from 2.0610 3 cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes. The blots were incubated with 1:2000 diluted mouse anti-HA antibodies (16B12; Covance, Berkeley, CA) in a blocking solution (1% bovine serum albumin, 1% nonfat dry milk, and 0.1% Tween 20 in phosphate-buffered saline), followed by a 1:10000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (Zymed Laboratories Inc., South San Francisco, CA) in a blocking solution. The membranes were then washed four times in 10 mM Tris-buffered saline with 0.1% Tween 20. The bound antibodies were visualized using enhanced chemiluminescence reagents (PerkinElmer Life Sciences, Boston, MA, USA) [26].

Construction of HA-ZFR1 overexpression strains
To generate the HA-ZFR1 overexpression construct, ZFR1 cDNA was first amplified by PCR using the primers OE-FW and OE-RV (Table S1), then inserted into the pBS-HA plasmid in such a way that expression of HA-ZFR1 was controlled by the MTT1 promoter. pBS-HA-ZFR1 was digested with SacI, and introduced into the CU428 and B2086 strains using the Biolistic PDS-1000/He particle-delivery system (Bio-Rad). The transformants were selected based on paromomycin resistance. The MTT1 replacement was analyzed by PCR with a pair of specific primers (MTT1-FW and MTT1-RV) (Table S1). Then, HA-ZFR1 over-expression was analyzed by real-time quantitative PCR using primers qRT-Pup and qRT-Pdown (Table S1). The following parameters were used for the PCR: 10 min at 95uC, followed by 40 cycles of 95uC for 15 s, 53 uC for 30 s, and 68uC for 35 s. A melting curve of the PCR products (60-90uC) was also obtained to ensure absence of artifacts. The values were normalized to the expression of the ribosomal 17S rRNA as an internal control.

Conjugation junction stability assay
After 3 h of mating, 200 ml of mating cells of wild type and ZFR1 knockout strains were collected and placed in 1.5 ml Eppendorf tubes. The samples were shaken on a vibrator at 8000 rpm for 5 min. Then, 20 ml of 20% trichloroacetic acid was added, and the numbers of paired and single cells were counted. Sampling of the cells was repeated three times.

Construction of truncated ZFR1 strains
The 59-truncated-ZFR1, 39-truncated-ZFR1, and 39, 59-truncated-ZFR1 genes were amplified by PCR using the specific primers truncated-59-FW and truncated-59-RV, truncated-39-FW and truncated-39-RV, and 59BamH1-T-FW and 39Asc1-T-RV (Table S1), respectively. The PCR products were then digested with BamH1 and Asc1 before cloning into a pBS-HA vector digested with BamH1 and Asc1. CU428 and B2086 cells were transformed with these constructs using the Biolistic PDS-1000/ He particle-delivery system (Bio-Rad Laboratories, USA). Brefeldin A Brefeldin A was added to cells at a concentration of 10 mg/ml. Cultures were incubated at 30uC for 3 h and CdCl 2 was added at 0.2 ug/ml after 2 h. Then, cells were starved and mated. Mating cells at 6 h postmixing were collected and examined for the localization of HA-tagged Zfr1p [14].

Characterization of ZFR1
ZFR1 was first identified in the Tetrahymena macronuclear genome database (http://www.ciliate.org), confirmed by PCR, then sequenced (data not shown). ZFR1 is 1347 bp long and consists of four exons that encode a predicted protein of 448 amino acids. The deduced protein sequence of Zfr1p contains an Nterminal B-Box zinc finger domain, and a C-terminal hydrophobic region (Fig. 1A). qRT-PCR analysis showed that ZFR1 was not expressed during the vegetative growth and starvation stages but was expressed specifically during the conjugation stages. In particular, the expression was up-regulated 2 h into the conjuga-  tion stage (Fig. 1B). This result is consistent with the expression profile of ZFR1 revealed by microarray data (Fig. S1) [19]. ZFR1 mRNA abundance was found to peak during the conjugation stage, implying that ZFR1 may play an important role in the process. It is well known that genes encoding proteins known to interact or to function in complexes show similar expression patterns, coordinate expression with putative genes of known function can identify genes with related functions [19]. To identify the signalling pathway by which Zfr1p functions, four genes coexpressed with Zfr1p were identified using the TGED database (http://tged.ihb.ac.cn/search.aspx?keyword = TTHERM_01285910) (Fig. S1) [19]. Of those, we found that TLR1 (TTHERM_00408910 encodes a transmembrane protein and TDT1 (TTHERM_00335970) encodes delta tubulin (correlation coefficient of co-expression with ZFR1 R = 0.987 and R = 0.948, respectively). Moreover, two new zinc finger domain containing genes ZFR2 (TTHERM_00637350) and ZFR3 (TTHERM_00531890) (correlation coefficient of co-expression with ZFR1 R = 0.999,0.869,respectively) were also identified. A gene network is useful to identify the genes involved in the same pathway, in a protein complex or that are co-regulated. Furthermore, Tetrahymena gene network (TGN) also indicated that ZFR1 is co-expressed with TLR1, TDT1, ZFR2 and ZFR3 [28].

ZFR1 is not essential in vegetative cells
To study the function of ZFR1, the open reading frame (ORF) of ZFR1 in the polyploid macronucleus was replaced by paromomycin resistance gene by homologous recombination (Fig. 2A). Replacement of the ZFR1 ORF was verified by PCR in five independent somatic knockout strains using a pair of specific primers (ZFR1-Fi and ZFR1-Ri). A single band, whose size was consistent with the size of the Neo4 cassette fragment, was amplified (Fig. 2B). To further confirm ZFR1 complete somatic knockout of ZFR1, different ZFR1 knockout cells, namely, gZFR1-C4 and gZFR1-B2, were mated. qRT-PCR analysis showed that no ZFR1 transcripts were produced before new macronuclei formed, but ZFR1 expression was restored when the new macronucleus formed after 8 h postmixing (Fig. 2C). This result showed that the parentally somatic macronuclear ZFR1 gene was completely replaced. The ZFR1 somatic knockout strains showed no obvious defects during vegetative growth (data not shown). This result was expected since ZFR1 is not expressed in growing and starved cells.

Zfr1p is required for conjugation development
To analyze Zfr1p function during conjugation, the developmental profiles of wild-type and ZFR1 knockout cells were compared ( Fig. 3A and 3B). At the initiation stage, there was no distinct difference between wild-type and ZFR1 knockout cells.  During the 7-8 h conjugation stage, about 20% of the cells were single and 80% of them had mating pairs. Unexpectedly, the proportion of single cells abruptly increased to 80% between 9-10 h. These separated single cells contained 5, 4, 3 or 2 micronuclei (Fig. S2). It seemed that the developing pairs were separated abnormally. If these separated cells were true progenies, they would have survived in the SPP medium containing 6methylpurine. The results showed that ,80% of the single cells were 6-methylpurine sensitive cells (Table 1). Hence, the surviving cells were not true sexual progenies. This indicated that normal development was aborted in ZFR1 knockout cells.
To confirm that developmental abortion was the real phenotype of ZFR1 disruption, ZFR1 knockout cells were mated with wildtype cells (Fig. 3C). Our results showed that conjugation development was rescued in these cells. It is well known that proteins can be transferred via the conjugation junctions between mating Tetrahymena cells [26,29,30]. It is possible that Zfr1p was also exchanged between wild-type cells and ZFR1 knockout cells in our experiments. Taken together, our results suggest that somatically expressed Zfr1p is necessary for conjugation development.

Localization of Zfr1p
To observe the localization patterns of Zfr1p, endogenous ZFR1 was replaced with HA-ZFR1 that encodes Zfr1p tagged with HA at the N-terminus (Fig. 4A). Endogenous ZFR1 in the macronucleus was partially replaced by HA-ZFR1 (Fig. 4B). HA-Zfr1p expression was examined by Western blot analysis using an anti-HA antibody (Fig. 4C). A single ,52 kDa band was observed, consistent with the predicted molecular weight of HA-Zfr1p (52.7 kDa). The expression profile of HA-Zfr1p was consistent with the mRNA expression profile of ZFR1. Development of HA-ZFR1 cells was similar to that of wild-type cells (data not shown), indicating that HA-Zfr1p was functional and reflected the function and distribution of endogenous Zfr1p.
Anti-HA staining was used to study the localization of HA-Zfr1p during the mating of different HA-ZFR1 cell types (HA-ZFR1-B5 x HA-ZFR1-C6). Specific localization of HA-Zfr1p did not occur when the mating was initiated (Fig. 4D-a). However, during conjugation development, HA-Zfr1p was localized in the conjugation junction when micronuclei began to elongate ( Fig. 4D-b). HA-Zfr1p was then constantly localized in the conjugation junction until pair separation occurred (Figs. 4D-c and 4D-d). HA-Zfr1p signal was no longer observed when the mating pairs were separated (Fig. 4D-e). Thus, HA-Zfr1p decorated the conjugation junction throughout the conjugation stage.

Overexpression of HA-Zfr1p
As described above, Zfr1p disruption resulted in the abortion of conjugation development and Zfr1p specifically decorated conjugation junctions. To further explore Zfr1p function, Zfr1p was over-expressed in Tetrahymena cells using an over-expression plasmid pBS-HA-ZFR1, in which ZFR1 was under the control of the MTT1 promoter (Fig. 5A). Two different mating types of overexpressed strains, namely OE-ZFR1-C3 and OE-ZFR1-B1, were obtained. Over-expression of HA-ZFR1 was detected by qRT-PCR and western blotting (Fig. 5B). Conjugation development of cells over-expressing HA-ZFR1 was comparable to that of wildtype cells (data not shown). Additionally, over-expressed HA-Zfr1p was specifically localized at conjugation junctions (Fig. 5C). This result further confirmed that Zfr1p specifically decorates conjugation junctions.
To understand the involvement of Zfr1p in the conjugation junction structure, a vibrating assay was performed at the early conjugation stage of mating cells. The results showed that mating pairs of the ZFR1 knockout cells were less stable than those of wildtype cells and ZFR1 overexpressed cells. In contrast, mating pairs of ZFR1 overexpressed cells were more stable than those of wildtype cells (Table 2). These results indicate that Zfr1p is involved in maintaining the stability of the conjugation junction during the early stages of conjugation.

Functional domain analysis of Zfr1p
Sequence analysis showed that Zfr1p has three characteristic structural domains (Fig. 6A). To explore the function of these domains, HA-tagged constructs in which either the N-terminal Bbox zinc finger domain (ZFR1 delN40 ), or the C-terminal hydrophobic domain (ZFR1 delC140 ), or both of these domains (ZFR1 delNC ) were deleted were made. Expression of these truncated genes was under the control of the MTT1 promoter. HA-Zfr1 delN40 (ZFR1 delN40 -B7.1 x ZFR1 delN40 -C8.1) and HA-Zfr1 delC140 (ZFR1 delC140 -B9.2 x ZFR1 delC140 -C10.3) were localized not only in the conjugation junction, but in linear rows of punctate foci throughout the cell (Fig. 6B-b and 6B-c). Interestingly, HA-Zfr1 delNC (ZFR1 delNC -B11.2 x ZFR1 delNC -C12.5) only formed linear rows of punctate foci throughout the mating cell (Fig. 6D). Hence, the N-terminal B-box zinc finger and C-terminal hydrophobic domains are both essential for proper localization of Zfr1p at the conjugation junction.
HA-Zfr1 delNC localized in ordered arrays near the plasma membranes of cells. This pattern was reminiscent of the arrangement of ciliary basal bodies, endosomes, Golgi apparatus and associated cortical mitochondria in Tetrahymena cells. To distinguish the localization of HA-Zfr1 delNC , dual labeling with antibodies against HA and centrin was conducted. Our results revealed that the truncated Zfr1p protein was localized near the basal bodies of cilia (Fig. 7). Golgi apparatus and associated cortical mitochondria localize to sites near basal bodies in Tetrahymena [31]. To further identify HA-Zfr1 delNC location, Brefeldin A was used. Brefeldin A is known to be a potent inhibitor of protein trafficking in the endomembrane system of cells. To test whether or not HA-Zfr1p trafficking is dependent on the Golgi apparatus, we compared HA-Zfr1p localization in cells treated with Brefeldin A and untreated cells. Brefeldin A treatment did indeed disrupt HA-Zfr1p localization at the conjugation junction in these cells, resulting in localization in ordered arrays near the plasma membrane (Fig. 8). This localization pattern of Zfr1p was similar to the localization of truncated Zfr1p.

Discussion
The model eukaryote T. thermophila is the first ciliated protozoan whose genome has been sequenced and whose genome-wide expression profile has been catalogued [18,19]. From the Macronuclear genome of this organism and subsequent analysis through comparative genomic hybridization, 24,725 proteincoding genes have been predicted [32]. Based on published microarray data for T. thermophilia, 1068 genes were identified, which are specifically expressed during the conjugation stage [19]. There are clusters of genes that exhibit distinct patterns of expression, which can be used to identify candidate genes involved in the striking developmental changes that occur during conjugation [19]. TWI1, which encodes an essential argonaute family Twi1p that is required for IES sequences elimination [26]. Four proteins (CnjBp, Wag1p, Ema1p and Giw1p) have been shown to be physically associated with Twi1p by co-immunoprecipitation and TAP-tagging [33][34][35]. Twi1p was also identified from isolated exchange junction preparation [12]. Similarly, the signal was detected at the junction of cells using anti-Ema1p antiserum [34]. So, Ema1p and Twi1p not only function in DNA elimination, they could also be involved in function of conjugation junction. In the study, we sought to identify new candidate functional genes associated with the conjugation junction in Tetrahymena.
ZFR1 is specifically expressed at conjugation stage. The expression patterns of ZFR1 imply Zfr1p play an important role in the stage. Immunofluorescence staining showed Zfr1p specifically localized at the conjugation junction. Although Zfr1p has no identified homolog in other organisms, it includes a zinc finger structure and a hydrophobic C-terminal domain. Zinc finger domains were first identified as DNA-binding motifs in the transcription factor TFIIIA in Xenopus laevis [36]. Now, they are recognized to bind DNA, RNA, protein and/or lipid substrates [37]. Zinc finger-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organization, zinc sensing, protein folding, chromatin remodeling, and cell adhesion [15]. In some cases, zinc fingers have been found to be crucial for the subcellular localization of proteins. For instance, the GTPase-activating protein ARF1 is localized to the Golgi complex via its zinc finger-like domains [38] and the autoantigen EEA1 is localized to endosomes via its zinc-binding FYVE finger [39]. In the present study, we found that the zinc finger domain of Zfr1p was required for Zfr1p specific localization at conjugation junctions. Truncated Zfr1p delN40 localized not only in the conjugation junction, but decorated Golgi apparatus.
It is well known that conjugation junction formation involves intricate interactions between membrane proteins, cytoskeletal frameworks, and lipids [8]. The cytoplasm near the conjugation junction contains numerous assorted membrane structures. These structures include lamellae, tubes, and small vesicles. Cda13p, containing membrane-spanning domains, is associated with the Golgi apparatus. When GFP-Cda13p was expressed in mating cells, a transient pattern of localization in which the fusion protein appeared to decorate a ring associated with the nuclear exchange junction was observed. Further studies have shown that Cda13p participates in events associated with remodeling of the nuclear exchange junction during Tetrahymena conjugation stage [14]. In the study, we found C-terminal hydrophobic domain of Zfr1p was also necessary for its specific conjugation junction localization. Furthermore, when the N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were disrupted, localization of Zfr1 delNC at conjugation junctions completely disappeared. So, it is clear that the C-terminal hydrophobic and N-terminal zinc finger domains of Zfr1p mediate interactions between Zfr1p and conjugation junction membrane structures or proteins. Accompanying truncated Zfr1 delNC disappeared at the conjugation junction, it occured at cortical puncta in longitudinal rows. It is well known that basal body, mitochondria, Golgi apparatus and endosomes are arranged in longitudinal rows in Tetrahymena [14,20,31]. Based on their different function, the Golgi apparatus is integral in modifying, sorting, and packaging macromolecules for secretion or use within the cell. To explore the relation between Zfr1p and Golgi apparatus, Brefeldin A, which inhibits protein trafficking in the endomembrane system of cells, was used. We found HA-Zfr1p localization in cells treated with Brefeldin A to be similar to the localization of truncated Zfr1p in untreated cells. Taken together, our results suggest that Zfr1p is transferred to conjugation junctions by Golgi apparatus and decorates the conjugation junctions via the N-terminal B-box zinc finger and C-terminal hydrophobic domains.
Originally, 15 proteins were identified from exchange junction preparations isolated via ethanol fixation and sonication. Using the method, many membrane proteins and proteins that are only loosely associated with the nuclear exchange junctions were lost. In addition, the identified 15 proteins were not conjugation junction specific. Fenestrin was seen over the oral apparatus, oral primordium, and the base of each cilium within the longitudinal ciliary rows and nuclear exchange junction [12]. Recently, it has been found that b-tubulin multigene family member BLT1 participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. BLT1 also decorated conjugation junction [40]. Ema1p localizes in old and new Macs during conjugation. Conjugation junction was also observed using anti-Ema1p antiserum [34]. GFP-Cda13p was localized along cortical rows and in cytoplasmic puncta. When GFP-Cda13p was expressed in mating cells, it also decorated a ring associated with the nuclear exchange junction [14]. So far, we found Zfr1p was only protein which specifically localized on the conjugation junction. So, we think more unidentified conjugation junction specific factors remain to explore. Although co-expressed genes in TGED and the network in TFGD are unavoidable to have some false positives, they are indeed useful to identify genes with related functions [19]. Based on the similar expression pattern and functional domains, four ZFR1 co-expressed genes, TLR1, TDT1, ZFR2 and ZFR3, were also identified using the TGED database and the Tetrahymena gene network (TGN) [19,28]. When ZFR2 or ZFR3 was knocked out, mating cells showed abnormal development phenotype which is similar to that of ZFR1 (data not shown). These results imply that these candidated co-expressed genes could be involved in the Zfr1p conjugation junction signal pathway. The physical and functional interaction of these co-expressed genes warrants further investigation.
Development of mating ZFR1 knockout cells looked normal at early stages. However, the conjugation junction structures between ZFR1 knockout cells were less stable than those of wild-type and ZFR1 over-expressing cells. These results implied the involvement of Zfr1p in conjugation junction structure stability. Importantly, an elaborate conjugation junction is required for pronuclear exchange during the conjugation stage in Tetrahymena [13]. The process of pronuclear exchange involves dramatic membrane remodeling to accompany the formation and resolution of the nuclear exchange junction during mating [14]. Evidence of abnormal development of ZFR1 knockout cells appeared 8 h into the conjugation stage, and true sexual progenies could not be produced. The separated single cells contained abnormal number of micronucleus (Fig. S2). These results indicate that pronuclear exchange or conjugation junction remodeling was aborted in ZFR1 knockout cells, which lead to abortion of sexual development.
Taken together, our study showed that ZFR1 is conjugation specific expressed protein. Zfr1p is first identified protein which only localized on the conjugation junction. It is essential for the sexual life cycle of Tetrahymena cells. Further studies on the interaction network of this protein will help to understand the highly complex and ordered restructuring of the conjugation junction structure in Tetrahymena.