Abstract
The biogenesis of Symbiodinium symbiosome in the host cells of the sea anemone, Aiptasia pulchella, involves retention of ApRab5 on and exclusion of ApRab11 from the organelle. One predicted consequence of this differential Rab association is the constant membrane fusion of symbiosomes with endocytic vesicles in the absence of parallel membrane retrieval and the subsequent formation of spacious symbiosomes, which nevertheless, contradicts the common perception. To solve this discrepancy, we determined whether membrane fusion occurs between symbiosomes and endocytic vesicles, and whether ApRab11-independent recycling is involved in symbiosome biogenesis. By using the biotin–avidin detection system, we found evidence for symbiosome–endocytic vesicle fusion. Cloning and characterization of ApRab4, an A. pulchella homolog of Rab4, showed that ApRab4 is associated with both the early endocytic and the perinuclear recycling compartments, and its normal function is required for the organization of the recycling compartments. Immunostaining localized ApRab4 to the symbiosome membrane, partially overlapping with ApRab5-decorated microdomains. Significantly, a treatment that impaired Symbiodinium photosynthesis also abolished symbiosome association of ApRab4. Furthermore, ApRab4 was quickly recruited to newly formed phagosomes, but prolonged association only occurred in those harboring live zooxanthelllae. We propose that ApRab4 retention on the symbiosome is an essential part of the mechanism for the biogenesis of Symbiodinium symbiosome.
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Acknowledgments
We would like to thank the two anonymous reviewers whose helpful comments improved the manuscript. This work was supported by grants from the National Science Council, Taiwan, to both M.-C. Chen (NSC 95-2311-B-022-001 and NSC 96-2311-B-022- 001) and L.-S. Fang (NSC 95-2313-B-291-003 and NSC 95-2313-B-291-003).
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Ming-Cheng Hong and Yung-Sen Huang contributed equally to this work.
The ApRab4 DNA sequence reported in this paper has been deposited to GenBank under accession no. EU116937.
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Fig. S1
Western blot analysis of ApRab4 in A. pulchella protein samples. The rat anti-ApRab4 antiserum (1:500 dilution) was used in combination with a HRP-conjugated goat anti-rat IgG secondary antibody (1:10,000 dilution) to detect ApRab4 in A. pulchella protein samples. a T 20 μg of total A. pulchella protein, R affinity-purified recombinant histine-tagged ApRab4 protein (20 ng). The recombinant protein contains additional 34 amino acid residues than its parental, wild-type protein. b M microsomal protein fraction, C cytosolic protein fraction of the tissue lysate of the animal. Protein fractions were prepared as described in “Materials and Methods”, and equal volumes of each protein extracts were fractionated in 15% SDS-PAGE gel. Positive protein bands were visualized by chemiluminescence using the Renaissance Western blot reagent from NEN. (GIF 23 kb)
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Hong, MC., Huang, YS., Song, PC. et al. Cloning and Characterization of ApRab4, a Recycling Rab Protein of Aiptasia pulchella, and Its Implication in the Symbiosome Biogenesis. Mar Biotechnol 11, 771–785 (2009). https://doi.org/10.1007/s10126-009-9193-2
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DOI: https://doi.org/10.1007/s10126-009-9193-2