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.