Alignment of ApRab4 and ApRab14-L proteins with

3. Results

3.2. Alignment of ApRab4 and ApRab14-L proteins with

Blastp search of the GenBank database showed that the deduced amino acid sequences of these cDNAs were highly homologous to those of known Rab4 and Rab14 proteins, with the highest overall sequence identity (87%

and 88%) to human Rab4 and Rab14 proteins, respectively. Multi- alignment analysis further revealed the extensive homology ApRab4 and ApRab14-L with Rab4 and Rab14 proteins from several model organisms, indicating that these proteins share similar three-dimensional conformation (Fig. 4, 5). In additional, sequence features shared by all GTP-binding proteins, in particular, the members of the Rab family, are all present on the deduced protein, including the four functional motifs involved in GTP-binding (G1–G4), the five Rab- specific motifs (RabF1–5), and a double cysteine (CPANC)- directed of ApRab4 and (CNC)-directed prenylation of ApRab14-L signal at

the C-terminal end （Stenmark and Olkkonen, 2001；Pereira-Leal and Seabra, 2000- 2001）. When the same set of Rab4 and Rab14 proteins sequences were subjected to phylogenetic analysis, ApRab4 and ApRab14-L were found to be more closely related to Xenopus tropicalis (frog) and Caenorhabditis elegans (worm), respectively (Fig. 6).

3.3. Characterized subcellular localization of EGFP-ApRab4 and EGFP-ApRab14-L in HeLa cells

To determine whether the homology of ApRab4 and ApRab14-L to others Rab4 and Rab14 proteins would extend to the functional level, I investigated the intracellular localization of ApRab4 and ApRab14-L. To this end, I constructed a series of recombinant plasmids encoding either EGFP-tagged ApRab4, ApRab14-L protein (pEGFP-ApRab4, pEGFP-ApRab14-L), or their mutants (including pEGFP- ApRab4-S22N,

pEGFP-ApRab4-Q67L, pEGFP-ApRab14-L- S26N, and

pEGFP-ApRab14-L-Q71L) and expressed them in HeLa cells. The G1 (Ser/Thr to Asn) mutants of ApRab4 and ApRab14-L (ApRab4-S22N and pEGFP-ApRab14-L-S26N) are likely to retain an inactive, GDP-bound conformation, while their G2 (Gln to Leu) mutants (pEGFP-ApRab4-Q67L and pEGFP-ApRab14-L-Q71L) are supposed to be in constitutively active, GTP-bound conformation. As shown in Fig. 8, EGFP fluorescence of GTP-bound ApRab4 was mainly associated with vesicular structures of

irregular sizes dispersed throughout the cytoplasm. On the contrary, GDP-bound ApRab4 displayed a diffusive distribution pattern in the cytoplasm. With regard to ApRab14-L, according to EGFP fluorescence, GTP-bound ApRab14-L not only localized on the irregular vesicular structures throughout the cytoplasm, but also accumulated at one side of the nucleus. Finally, GDP-bound ApRab14-L showed strong tendency to cluster around the nucleus.

3.4. Characterized of EGFP-ApRab4 labeled structures

To further identify the localization of ApRab4 on endocytic pathway, HeLa cells were transfected with the plasmid encoding EGFP-ApRab4.

Different species of endocytic compartments were labeled with Texas red-conjugated transferrin（Ghosh and Maxfield, 1995）, TRITC-conjugated dextran, or LysoTracker, by the procedure described under “Materials and methods”. Co-localization of ApRab4 with dextran was apparent as indicated by strong overlapping signals after labeling for 10 minutes but significantly reduced when the labeling was prolonged for 30 minutes (Fig. 10). This result suggested that ApRab4 localizes preferentially to early endosomes. When the degree of co-localization between transferrin, and EGFP-ApRab4 signals were examined, high levels of co-localization were noted when HeLa cells were labeled with transferrin for 30 minutes to saturate all recycling endosomes (Fig. 9). This result suggested that ApRab4 also localizes to

recycling endosomes. Finally, as shown in Fig. 12, there was no overlapping between the green fluorescence signals of EGFP-ApRab4 and the red fluorescence signals of LysoTracker, which indicated that ApRab4-positive vesicles are not acidic compartments. Overall, the results of my study suggested ApRab4 is most likely present on both early endosomes and transferrin-positive recycling endosomes.

3.5. C h a r a c t e r i z e d of EGFP–ApRab14-L labeled structures

To determine the identities of EGFP-ApRab14-L -positive structures and the role of ApRab14-L in the trafficking between trans-Golgi network and early endosomes, HeLa cells were first trasfected with the plasmid encoding EGFP-tagged ApRab14-L, and then incubated with TRITC- dextran, transferrin-Texas Red, LysoTracker, or TR-C⁵- ceramide to specifically label early, late endosomes, recycling endosomes, lysosomes and the Golgi apparatus. As shown in Fig. 9 and 11, EGFP signals of GTP-bound ApRab14-L and ApRab14-L were highly co-localized with the structures labeled by 10-min incubation of either dextran or transferrin. Such a high degree of co-localization gradually disappeared when the incubation period was prolonged to 30 minutes (Fig. 9.11). When ApRab14-expressing HeLa cells were stained with TR-C⁵-ceramide to label the Golgi apparatus, it was found that GDP-bound ApRab14 displayed a very extensive co-distribution with the Golgi apparatus, whereas the wild type ApRab14 only partially

overlapped with the red TR-C⁵- ceramide signals (Fig. 13). Finally, as observed for ApRab4, there was no overlap between the red signals of LysoTracker and the green signals of EGFP-ApRab14 (Fig. 12). Overall, the results of my study suggested that EGFP-ApRab14 protein was present on both early endosomes and the Golgi apparatus.

3.6. C h a r a c t e r i z e d of antibodies by western blot analysis

To investigate the potential involvement of ApRab4 and ApRab14-L in the regulation of phagosome maturation in Aiptasia phagocytic cells, two specific rat polyclonal antisera, one specific for ApRab4, the other for ApRab14, were prepared as described in the ”Materials and Methods” using the corresponding affinity-purified histidine-tagged ApRab proteins as the immunogens, and used as specific probes for the detection of endogenous ApRab4 and ApRab14-L proteins, respectively. To determine whether these antibodies can specifically recognize ApRab4 and ApRab14-L in highly complex protein mixtures, I prepared and analyzed total Aiptasia tissue homogenates by Western blot using the rat anti-ApRab4 and -ApRab14-L antisera as the primary antibodies. As revealed in Fig. 7, the rat anti-ApRab4 antiserum recognized a single protein band of approximately 24 kDa in size, which is in close agreement with the expected size of ApRab4. On the other hand, the rat anti-ApRab14-L antiserum strongly reacted with two protein bands of approximately 24 kDa and 8 kDa in size, both of which are

consistent with the calculated sizes of ApRab14-L and ApRab14-S, respectively. Together, the results of my Western blotting analyses strongly suggested that the two antisera I have prepared are capable of specifically detect their corresponding antigens, and therefore are suitable for immunocytochemical studies, which I described below.

3.7. Localized of endogenous ApRab4 and ApRab14-L in the host cells of zooxanthellae by immunofluorescence analysis

To examine whether ApRab4 and ApRab14-L are involved in the maintenance of endosymbiotic association of zooxanthellae with their host sea anemones, symbiotic A. pulchella was processed for ApRab4 and ApRab14-L immunofluorescence analysis (Fig. 14) with or without prior treatment of the photosynthesis inhibitor, DCMU, and the percentages of symbiosomes (phagosomes containing resident zooxanthellae) associating with either ApRab4 or ApRab14-L were then determined. In control animals (0-h DCMU treatment), a major proportion of symbiosomes were found to possess detectable ApRab4 (84%) and ApRab14-L (76%) staining signals, and a sharp decrease (more than four folds) in the positive counts of either ApRab4- or ApRab14-L- associated symbiosomes were obtained after only 1-h DCMU treatment (Fig. 16). This finding of my study demonstrated clearly that the association of ApRab4 and ApRab14-L with the symbiosomes is brought about by a yet unknown effect exerted by the healthy

zooxanthellae dwelling inside.

3.8. Involvement of ApRab4 and ApRab14-L in Phagocytosis

To investigate the potential involvement of ApRab4 and ApRab14-L in the establishment and/or maintenance of the endosymbiotic association of zooxanthellae with their native animal host A. pulchella, immunofluorescence analysis was employed to examine the association of ApRab4 and ApRab14-L with the phagosomes containing newly internalized latex bead.

As shown in Fig. 15, ApRab4 and ApRab14-L staining signals were apparently associated with latex bead-containing phagosomes. Noticeably, these signals were distributed as discrete patches around the phagosomes. As short as 15 minutes after phagocytosis, approximately 74% and 80% of phagosomes containing latex beads were already associated with ApRab4 and ApRab14-L, respectively. The percentage of ApRab4- or ApRab14-L-positive phagosomes gradually decreased to respective 16% and 12% of the phagosome population at the end of the phagocytosis assays (45 minutes) (Fig. 17). These results strongly suggested that both ApRab4 and ApRab14 preferentially associate with early phagosomes, and therefore are likely participating in the early phases of phagosomes maturation through regulating vesicle budding from the maturing phagosomes. On another phagocytosis assay by feeding live or heat-killed zooxanthellae, 50% and 60% of 15 minutes old phagosomes containing heat-killed zooxanthellae were

positive for ApRab4 and ApRab14-L signals. They were decreased to 20%

and 50% in 30-minutes old phagosomes and further down to 15% and 10% in 45-minutes old phagosomes as shown in Fig. 18. For feeding with live zooxanthellae, there are 45% and 55% of 15-minutes-old phagosome population which were detected ApRab4- and ApRab14-L-signals around phagosomes. But not decreased whether on 30-minutes-old phagosomes or 45-minutes-old phagosomes, they were still highly retained on them, as shown in Fig. 19.

4. Conclusion and Discussion

In this work, cDNAs for ApRab4 and two isoforms of ApRab14 have been successfully cloned and partially characterized. (Fig. 1-3). These full-length cDNA sequences contain 5’ and 3’ UTR, start and stop codon, poly (A) tails, Rab family specific conserved domains and double cysteine at 3’ end of coding region (except ApRab14-S had no double cysteine).

Multi-alignment analysis of ApRab4 and ApRab14-L indicated that they are highly homologues with human Rab4 and Rab14. Therefore, ApRab4 and ApRab14-L were belonged to family of Rab protein (Fig. 4-6).

In my attempt to clone the A. pulchella homologue to ApRab14, two ApRab14-like sequences were detected. The longer one, ApRab14-L, is 654 bp in length, while the shorter one, ApRab14-S, 228 bp. The amino acid sequence of ApRab14-S was essentially identical to that of ApRab14-L N-terminus with two exceptions. The first one occurred at the 51th amino acid (Thr/Pro) and the second being the last 5 amino acids of ApRab14-S.

Because of the 51th amino acid alteration, the folding of ApRab4-S is probably changed to a significant level. In addition, since ApRab14-S had no double cysteine at its C-terminus, subsequently prenylation is not likely. I proposed that ApRab14-S may function by interacting with ApRab14-L.

According to the EGFP report analysis described above, ApRab4 was present on the transferrin-positive structures (Fig. 9) and early endosomes

(Fig. 10). These results indicate that EGFP-ApRab4-decorated structures are indeed recycling endosomes, and consequently, normal endocytic recycling will likely be inhibited by GDP-bound ApRab4. Examination in EGFP-ApRab14-L expressing cells showed that ApRab14-L was present on dextran-, transferrin-, or TR-C⁵-ceramide-positive structures (Fig. 9, 11, 13).

And, when GDP-bound EGFP-ApRab14-L was expressed in HeLa cells, the green fluorescence mostly overlapped with TR-C⁵- ceramide staining signals.

This result suggested that ApRab14 in its GDP-bound state will locate on the Golgi apparatus (Fig. 13). All results suggested that ApRab14-L participate in the recycling pathway and membrane trafficking between the Golgi apparatus and early endosomes. The intracellular localization patterns of ApRab4 and ApRab14-L that I have observed are consistent with previous mammalian studies, and therefore, their function may be similar to their mammalian homologues.

To determine whether or not the rat anti-ApRab4 and the rat anti-ApRab14-L antiserum that I have prepared can specifically recognize ApRab4 and ApRab14-L in total Aiptasia tissue homogenates, Western blot analysis was executed, as shown in Fig. 7. A major band approximately 24 kDa in size was detected by the rat anti-ApRab4 antiserum, which was close to the calculated molecular weight of ApRab4 protein. Western blot by using the rat anti- ApRab14-L antiserum revealed two major bands. One was approximately 24 kDa and the other was 8 kDa in size, which were in close

agreement with the calculated molecular weight of ApRab14-L and ApRab14-S proteins, respectively. Therefore, it was concluded that the two antisera were able to specifically recognize the three intended proteins in

Aiptasia

total protein preparation. Although the rat anti-ApRab14-L polyclonal antibody reacted both with ApRab14-S and ApRab14-L, but sequence analysis of ApRab14-S protein was shown there are no double cysteine in its C-terminus. All previous studies report that all Rabs require a double C-terminus cysteine to anchor to their target membrane (Casey and Seabra, 1996). So I proposed ApRab14-S protein can not anchor to membrane by itself. The signals detected on organelle membrane by the rat anti-ApRab14-L polyclonal antibody could come from two potential sources, one from ApRab14-L, and the other from the complex between ApRab14-S and ApRab14-L. But no research has reported that Rabs can interact with each other and form a complex. Therefore, there is high probability that the signals detected on organelle membrane are from ApRab14-L protein.

Accordingly, it should be appropriate to use the rat anti-ApRab14-L antibody be in immunofluorescence assays to detect ApRab14-L protein.

In my phagocytosis assays (Fig.17, 18, 19), phagosomes containing newly acquired dead algae seemed to follow a maturation path similar to that taken by phagosomes containing latex beads—quick acquisition of and then gradual removal of ApRab4 and ApRab14-L from phagosome membrane, except that the former appeared to develop at a bit faster pace. A different

picture emerged when ApRab4 or ApRab14-L association with phagosomes containing newly internalized live zooxanthellae was examined. Although a general decreasing trend was followed, significantly lower percentages of these phagosomes were positive for ApRab4 and ApRab14-L. In all cases, the positive counts (in percentage) of ApRab4- and ApRab14-L-associated phagosomes containing newly internalized live zooxanthellae ranged from a mere quarter to one-half of those for the heat-killed zooxanthellae-containing counterparts. These results were shown that ApRab4 and ApRab14-L were maintained in early stage of phagosome maturation and indicated normal phagosome maturation pathway was interfered by live zooxanthellae. That‘s a reason which ApRab4 and ApRab14-L protein is discovered on the symbiosomes (modified phagosomes by live zooxanthellae), but not on the phagosomes containing dead zooxanthellae.

Results of phagocytosis assays and immunofluorescence assay showed that ApRab4 and ApRab14-L were likely involved in early stage of phagosome maturation and were present on the zooxanthellae-housing symbiosomes in Aiptasia digestive cells. The combined results described above suggested that ApRab4 and ApRab14-L function on

Aiptasia-Symbiodinium endosymbiosis and are involved in the regulation of

endocytic recycling and vesicle transport between symbiosomes and the Golgi apparatus of Aiptasia host cells. Overall, the results of above experiments strongly suggest that both ApRab4 and ApRab14-L play critical

roles in the endocytic recycling pathway and the biogenesis of symbiosomes in the Aiptasia-Symbiodinium endosymbiosis.

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6. Figures

Fig. 1.

Primary sequences of A. pulchella Rab4 (ApRab4). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.

ApRab14-L full length cDNA

Fig. 2.

Primary sequences of A. pulchella Rab14-L (ApRab14-L). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.

ApRab14-S full length cDNA

GGCCATT 7 ACGGCCGGGAATCACGAGATCCGCCATCTTTCCTAAAGTCTTCTT 52 CCGGATTTC TAATATAATTTAATATTCCCTTACCAGGTCGCAAAG 97 ATGGCAGCGACAGGGCCGTACAATTATTCCTATATATTCAAGTAT 142

M A A T G P Y N Y S Y I F K Y 1 5 ATTATTATAGGTGATATGGGTGTTGGTAAGTCATGTCTCCTTCAC 187 I I I G D M G V G K S C L L H 3 0 CAATTCACAGAAAAGAAATTCATGGCAGACTGCCCTCACACAATT 232 Q F T E K K F M A D C P H T I 4 5 GGTGTAGAGTTTGGCCCAAGAATAATAGAGGTAGCTGGACAAAAG 277 G V E F G P R I I E V A G Q K 6 0 ATCAAGCTTCAGATATGGGATACGGCAGGCTACTATTTATCTCCT 322 I K L Q I W D T A G Y Y L S P 7 5 TAAGCTGAATCAAAATTTACAAGTTAATGAAAAACATATTGAATT 367

CT CATTTGATTATAAAC T CCTTGCAGTTAGT CTCTTGTGT CTTCA 4 1 2 CATTGTAGCTAGATTAATGTGATGACACCGGAAATAGCTGCAAGG 457 AGCCTAACTGAACGAAGCTTAACTGTTGTTACAGTAAAATATATT 502 T G CT GT G GT TAAA AT TAC AT T GTAT T C AG G AT T T G AAT T T T T TT T 5 4 7 CAATAAGATAAG ACAG CTCTTCATTTGTTAAAATGGCTGATAAAC 5 92 TAC ACTAAT T GAC AGT GAAT GTAATTT GAATATT GT CATAGC AAC 6 37 CC AG AAT T CTACAT TTCC AT GAC AAC C ACT GNT GT CTATAAC ATT 68 2 GTCATGGTAACTTAGAAGAAGT 704

Fig. 3.

Primary sequences of A. pulchella Rab14-S (ApRab14-S). The start codon and a proceeding in-frame stop codon are in bold letters. The stop codon is indicated by an asterisk. The putative polyadenylation signal is underlined.

Fig. 4.

Multi-alignment of ApRab4 protein with selected Rab4 relatives.

Amino acid residues identical in all Rab4 proteins are shown on a black background; residues conserved in most members are shown on a gray background. Sequence motifs that are highly conserved among Rab proteins are indicated by solid lines and labeled. Functional motifs involved in

GTP-binding are marked with solid lines and numbered (G1-G4). Sequence motifs that are highly conserved in Rab family proteins are indicated by solid lines and labeled (RabF1–RabF5). The putative C-terminal double cysteine prenylation signal is underlined and labeled with the letter P. The GenBank accession numbers of the selected Rab4 proteins are: AAP97171 (human);

AAL11725 (mouse); AAH44974 (frog); NP 523777 (fly); NP 001004002 (fish); and NP 059051 (rat).

Fig. 5.

Multi-alignment of ApRab14-L protein with selected Rab14 relatives.

Amino acid residues identical in all Rab14 proteins are shown on a black background; residues conserved in most members are shown on a gray background. Sequence motifs that are highly conserved among Rab proteins are indicated by solid lines and labeled. Functional motifs involved in

在文檔中美麗海葵 Rab4 和 Rab14-L 蛋白在胞內共生所扮演的角色之研究 (頁 21-0)