Summary
A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, remain in the cell body. Thus, polarized PC12 cells enhance the resolution for immunofluorescence microscopic mapping of protein localization in the early biosynthetic pathway. The authors also describe here a rapid cell fractionation protocol employing velocity sedimentation in iodixanol gradients that allows one-step separation of the pre-Golgi vacuoles, tubules, and RER.
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Acknowledgments
The authors are grateful to Eivind Rødahl (Haukeland University Hospital, Bergen) for the kind gift of the PC12 cell subclone and Bruno Goud (Institut Curie, Paris) for providing the antiserum against Rab1. This work was financially supported by The University of Bergen, The Norwegian Cancer Society and The Functional Genomics (FUGE) Program of the Research Council of Norway.
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Sannerud, R., Michaël Marie, Hansen, B.B., Saraste, J. (2008). Use of Polarized PC12 Cells to Monitor Protein Localization in the Early Biosynthetic Pathway. In: Vancura, A. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 457. Humana Press. https://doi.org/10.1007/978-1-59745-261-8_19
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DOI: https://doi.org/10.1007/978-1-59745-261-8_19
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