Functional characterization of the partially purified Sac1p independent adenine nucleotide transport system (ANTS) from yeast endoplasmic reticulum

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

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Functional Characterization of ERp18, a New Endoplasmic Reticulum-located Thioredoxin Superfamily Member

Journal of Biological Chemistry ◽

10.1074/jbc.m304598200 ◽

2003 ◽

Vol 278 (31) ◽

pp. 28912-28920 ◽

Cited By ~ 61

Author(s):

Heli I. Alanen ◽

Richard A. Williamson ◽

Mark J. Howard ◽

Anna-Kaisa Lappi ◽

Heli P. Jäntti ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Functional Characterization

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Functional characterization of orchardgrass endoplasmic reticulum-resident Hsp90 (DgHsp90) as a chaperone and an ATPase

Plant Physiology and Biochemistry ◽

10.1016/j.plaphy.2009.06.008 ◽

2009 ◽

Vol 47 (10) ◽

pp. 859-866 ◽

Cited By ~ 11

Author(s):

Joon-Yung Cha ◽

Min Hee Jung ◽

Netty Ermawati ◽

Mukhamad Su'udi ◽

Gyu-Jin Rho ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Functional Characterization

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Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00309.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. G660-G668 ◽

Cited By ~ 67

Author(s):

Katsuhisa Inoue ◽

Yasuhiro Nakai ◽

Sayaka Ueda ◽

Shunsuke Kamigaso ◽

Kin-ya Ohta ◽

...

Keyword(s):

Small Intestine ◽

Transport System ◽

Functional Characterization ◽

Cellular Membrane ◽

Molecular Entity ◽

Xenopus Laevis Oocytes ◽

Rat Small Intestine ◽

Distribution Profile ◽

Folate Transport

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant ( Km) for the former (2.4 μM) than for the latter (5.7 μM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the Km values were 1.2 and 5.8 μM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.

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Expression of a Plastidic ATP/ADP Transporter Gene inEscherichia coliLeads to a Functional Adenine Nucleotide Transport System in the Bacterial Cytoplasmic Membrane

Journal of Biological Chemistry ◽

10.1074/jbc.273.16.9630 ◽

1998 ◽

Vol 273 (16) ◽

pp. 9630-9636 ◽

Cited By ~ 62

Author(s):

Joachim Tjaden ◽

Christian Schwöppe ◽

Torsten Möhlmann ◽

Paul W. Quick ◽

H. Ekkehard Neuhaus

Keyword(s):

Transport System ◽

Cytoplasmic Membrane ◽

Adenine Nucleotide ◽

Transporter Gene ◽

Adenine Nucleotide Transport

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Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

Molecular Biology of the Cell ◽

10.1091/mbc.4.9.931 ◽

1993 ◽

Vol 4 (9) ◽

pp. 931-939 ◽

Cited By ~ 52

Author(s):

D Feldheim ◽

K Yoshimura ◽

A Admon ◽

R Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Signal Sequence ◽

Functional Characterization ◽

Yeast Cells ◽

Temperature Sensitive ◽

Endoplasmic Reticulum Membrane ◽

Restrictive Temperature ◽

Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

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