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Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein

Nature Cell Biology volume 2, pages 125–127 (2000)Cite this article

In higher eukaryotes, secretory and plasma-membrane proteins are transported from the endoplasmic reticulum (ER) to a central Golgi complex and subsequently packaged into membrane-bound carriers for delivery to the cell surface. The long-distance transport of post-Golgi organelles to the tips of axons or to developing hyphal extensions in Neurospora crassa shows an absolute requirement for microtubules and microtubule-associated motors1,2. In contrast, microtubule disruption only moderately attenuates Golgi-to-plasma-membrane transport in fibroblasts and randomizes surface delivery of select proteins in epithelial cells3,4. The observed preservation of biosynthetic transport after microtubule disruption is probably due to the extensive fragmentation and redistribution of Golgi mini-stacks to regions immediately adjacent to both the ER and the plasma membrane3,5,6. Here we have designed experiments to test the hypothesis that when the characteristic central localization of the Golgi is preserved, microtubules, kinesin and the GTPase dynamin are essential for post-Golgi trafficking. We ruled out a pharmacological approach to tackling this problem because we found that microtubule antagonists caused dispersal of the Golgi complex before complete microtubule disassembly occurred (see Supplementary Information ). Instead, we microinjected function-blocking anti-kinesin antibodies HD and SUK-4 or cDNAs encoding a dominant-negative form of dynamin into cells expressing a green fluorescent protein (GFP)-tagged apical-membrane protein, p75. We show that kinesin and dynamin are required for different stages of post-Golgi transport.

To test the role of kinesin in post-Golgi transport, we sequentially injected anti-kinesin antibodies and p75–GFP cDNA into MDCK cells. The polyclonal antibody, HD, inhibits centrifugal movement of pigment granules7, inhibits pseudopodial activity8, and disrupts intermediate-filament networks9. The monoclonal antibody, SUK-4, inhibits pH-regulated extension of tubular lysosomes10. Neither HD nor SUK-4 IgG affected accumulation of p75–GFP in the Golgi (Fig. 1a, HD/SUK-4, 0 min). HD, but not SUK-4, blocked the redistribution of p75–GFP after shifting to 32 °C (Fig. 1a, HD/SUK-4, 240 min). As HD reacts with multiple kinesin-family members whereas SUK-4 is specific for conventional kinesin11, we conclude that a kinesin other than conventional kinesin is involved in post-Golgi trafficking of p75–GFP.

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Figure 1: Inhibition of kinesin activity or expression of D2K44E inhibits release of p75–GFP from the Golgi/TGN after release from a block at 20 °C.
Figure 2: Post-Golgi trafficking of p75–GFP-containing vesicles and tubules after release from a 20 °C temperature block is mediated by kinesin and dynamin.

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Acknowledgements

This work was supported in part by a grant from the NIH and by a Jules and Doris Stein professorship of the Research to Prevent Blindness Foundation (to E.R.-B.) and by an NIH NRSA fellowship (to G.K.).

Correspondence and requests for materials should be addressed to E.R.-B.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology.

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Author notes

  1. Alan Marmorstein

    Present address: Department of Ophthalmic Research, Cleveland Clinic Foundation, Cleveland, Ohio, 44195, USA

Authors and Affiliations

  1. Dyson Institute for Vision Research, Joan and Sanford Weill Medical College of Cornell University,, New York, 10021, , New York, USA

    Geri Kreitzer, Alan Marmorstein & Enrique Rodriguez-Boulan

  2. Department of Cell Biology, University of Massachusetts Medical School, Worcester, 01655, , Massachusetts, USA

    Patricia Okamoto & Richard Vallee

Authors
  1. Geri Kreitzer
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Correspondence to Enrique Rodriguez-Boulan.

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Kreitzer, G., Marmorstein, A., Okamoto, P. et al. Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein. Nat Cell Biol 2, 125–127 (2000). https://doi.org/10.1038/35000081

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