Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.
A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells
1. Abbreviations used in this paper: aa, amino acids; GH and GL, heavy and light Golgi fraction, respectively; PH, pleckstrin homology; RM and SM, rough and smooth microsomes, respectively; RT-PCR, reverse transcriptase–PCR.
We are especially grateful to Mrs. B. Oswald for the initial identification and isolation of DLP1, antibody purification, and other technical assistance. We thank Dr. S.L. Schmid for providing the antidynamin antibody, hudy 1; Dr. L. Traub for anti–γ-adaptin antibody; Dr. J.-P. Paccaud for anti-Sec23p; Dr. D.D. Sabatini for anti-Rab8; Dr. M.S. Robinson for anti-μ3; and Dr. N.F. LaRusso for anti–β-galactosidase antibody and the human cholangiocyte cell line. Cryosectioning of liver tissue by Mrs. J. Mui in Dr. J. Bergeron's laboratory at McGill University is gratefully acknowledged. We are also grateful to Mr. E. Krueger for helping with photographic techniques, and to Dr. J.R. Henley and Ms. R.R. Torgerson for helpful comments and reading the manuscript.
This study was supported by National Institutes of Health (NIH) training grant (DK07198) and National Research Service Award postdoctoral fellowship from National Institute of Diabetes and Digestive and Kidney Diseases (DK09574) to Y. Yoon and NIH grant (DK44650) to M.A. McNiven.
Address correspondence to Mark A. McNiven, Center for Basic Research in Digestive Diseases and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. Tel.: (507) 284-0683. Fax: (507) 284-0762. E-mail: [email protected]
Dr. Dahan's current address is Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905.
Yisang Yoon, Kelly R. Pitts, Sophie Dahan, Mark A. McNiven; A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells . J Cell Biol 23 February 1998; 140 (4): 779–793. doi: https://doi.org/10.1083/jcb.140.4.779
Download citation file:
Sign in
Client Account
Sign in via your Institution
Sign in via your InstitutionEmail alerts
Advertisement
Advertisement
