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The Rab8 GTPase regulates apical protein localization in intestinal cells

Abstract

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells1,2,3,4,5,6,7. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway8,9. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.

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Figure 1: Accumulation of apical but not basolateral proteins in cytoplasm of Rab8-deficient intestinal epithelial cells.
Figure 2: The reduction of apical markers in the small intestine of the knockout mice causes a reduction of substrate uptake from the apical side.
Figure 3: Microvillus atrophy and inclusions in Rab8-deficient mice.
Figure 4: RAB8 is greatly reduced in intestinal epithelial cells of the microvillus inclusion disease patient.

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Acknowledgements

We thank M. Takano, T. Horie, R. Hirai, Y. Okada, C. Ohsawa, M. Sugiura, H. Hata and R. Ishida for assistance with cell culture, uptake studies, animal care, and microscopy, H. Gomi, T. Izumi, M. Komachi, C. Mogi, H. Tomura, F. Okajima, N. Shihara and T. Suzuki for teaching us various techniques, Y. Wada, Y. Uchiyama, H. Homareda, K. Inui and Y. Umesaki for providing antibodies, J. Miyazaki for providing the transgenic mice, S. Mitani for providing rab-8 knockout worms, and Y. Ishida for providing the construct. We thank the affected individuals and their families for providing the blood samples. The Lamp2 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa. We also thank H.-P. Zimmer, A. Ballauff and T. Berger for providing DNA samples of the patients. This work was supported by grants-in-aid and the 21st century Center of Excellence Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology to T.S., M.S., K.S. and A.H.

Author Contributions T.S. generated and analysed Rab8 knockout mice. S.M. took care of the microvillus inclusion disease patient and collected the blood and small intestine samples. Y. Kato supervised the measurement of the absorption rate of nutrients in the intestine of the mice. K.S. and M.S. generated the transgenic C. elegans and photographed them. N.T. generated the chimeric mice. K.M. took care of the microvillus inclusion disease patient. K.O. supervised the treatment of the patient. Y. Kubo. measured the absorption rate of nutrients in the intestine. A.T. provided the instruments and supervised the absorption experiments. R.H. stained the tissue, arranged figures and wrote part of the manuscript. A.H. planned and supervised the experiments, performed morphological analyses, arranged figures and wrote the manuscript

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Sato, T., Mushiake, S., Kato, Y. et al. The Rab8 GTPase regulates apical protein localization in intestinal cells. Nature 448, 366–369 (2007). https://doi.org/10.1038/nature05929

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