A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

Abstract

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.

The Dynamin-like GTPase DLP1 Is Essential for Peroxisome Division and Is Recruited to Peroxisomes in Part by PEX11

“…Cells were then incubated at 37°C and processed for immunoblot and indirect immunofluorescence analyses at various times after transfection. DLP1 is known to have multiple isoforms that are generated by alternative splicing of the initial DLP1 transcript (20,33). Our immunoblot analysis indicated that at least two different isoforms were present in fibroblasts (Fig.…”

“…DLP1 is the human gene most similar to two of three dynamin-like proteins found in yeast, Vps1p and Dnm1p. DLP1 is also known to regulate the dynamics of mitochondria and the endoplasmic reticulum (20,21). We report here that a reduction in DLP1 activity inhibits peroxisome division and reduces peroxisome abundance.…”

“…However, the human DLP1 gene product, which is the human protein that shows the greatest sequence similarity with S. cerevisiae Vps1p, shares even greater similarity with S. cerevisiae Dnm1p, a yeast protein that regulates mitochondrial division and endosome dynamics but has no discernible role in peroxisome division (19,30). Furthermore, human DLP1 has been shown previously to regulate mitochondrion division and endoplasmic reticulum dynamics (20,21), indicating that it might share more functional similarity with Dnm1p than with Vps1p. Therefore, it was essential to empirically determine whether human DLP1 has a role in peroxisome biogenesis.…”

“…Antibodies-Two affinity-purified rabbit anti-DLP1 peptide antibodies anti-DLP1MID and anti-DLP1N were provided by Dr. McNiven (Mayo Clinic and Foundation, Rochester, MN) (20). Antiserum against the COOH-terminal 16 amino acids of DLP1 (anti-DLP1C) was generated by injecting rabbits with ovalbumin (Pierce) coupled peptide NH 2 -CKQGASQIIAEIRETHLW-COOH.…”

Peroxisome proliferator-activated receptor α-independent peroxisome proliferation

“…Each mRNA expression level was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression level, and subsequently normalized to that of wild-type mice fed a control diet. In the case of analysis of dynamin-like protein 1 (DLP1) mRNA, Northern blotting was performed as described previously [2] and hybridized with a 32 P-labeled rat cDNA probe [17] because the mouse cDNA sequence…”