Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p

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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Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells

Analytical Chemistry ◽

10.1021/acs.analchem.1c00893 ◽

2021 ◽

Author(s):

Christoph Gstöttner ◽

Tao Zhang ◽

Anja Resemann ◽

Sophia Ruben ◽

Stuart Pengelley ◽

...

Keyword(s):

Mammalian Cells ◽

Functional Characterization

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A proline-rich region from maize γ-zein adopts a left-handed amphipathic structure that may act as a signal for its retention in the endoplasmic reticulum

Peptides 1994 ◽

10.1007/978-94-011-1468-4_19 ◽

1995 ◽

pp. 60-61 ◽

Cited By ~ 2

Author(s):

I. Dalcol ◽

F. Rabanal ◽

F. Albericio ◽

M. Pons ◽

M. Geli ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Rich Region ◽

Left Handed ◽

Proline Rich Region

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Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

Molecular Biology of the Cell ◽

10.1091/mbc.e05-03-0267 ◽

2005 ◽

Vol 16 (10) ◽

pp. 4648-4659 ◽

Cited By ~ 132

Author(s):

Brandon E. Kremer ◽

Timothy Haystead ◽

Ian G. Macara

Keyword(s):

Mammalian Cells ◽

Spectroscopic Analysis ◽

Molecular Function ◽

Rich Region ◽

Intact Cells ◽

Pronounced Increase ◽

Binding Partner ◽

Microtubule Stability ◽

Proline Rich Region

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

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In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

Journal of Biological Rhythms ◽

10.1177/0748730416668898 ◽

2016 ◽

Vol 31 (6) ◽

pp. 540-550 ◽

Cited By ~ 18

Author(s):

Kevin A. Feeney ◽

Marrit Putker ◽

Marco Brancaccio ◽

John S. O’Neill

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Firefly Luciferase ◽

Luciferase Reporter ◽

Circadian Gene ◽

Cultured Fibroblasts ◽

High Concentrations ◽

Lower Temperature ◽

Kinetics Of

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

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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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The Ca2+-binding Protein ALG-2 Is Recruited to Endoplasmic Reticulum Exit Sites by Sec31A and Stabilizes the Localization of Sec31A

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0444 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4876-4887 ◽

Cited By ~ 72

Author(s):

Akinori Yamasaki ◽

Katsuko Tani ◽

Akitsugu Yamamoto ◽

Naomi Kitamura ◽

Masayuki Komada

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Binding Protein ◽

Small Gtpase ◽

Dependent Manner ◽

Rich Region ◽

Linked Gene ◽

Transport Vesicles ◽

Er Exit Sites ◽

A Cell

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca2+-dependent manner and colocalized with Sec31A at ER exit sites. A Ca2+binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca2+chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca2+-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

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Functional characterization of ecdysone receptor gene switches in mammalian cells

FEBS Journal ◽

10.1111/j.1742-4658.2006.05545.x ◽

2006 ◽

Vol 273 (24) ◽

pp. 5550-5563 ◽

Cited By ~ 8

Author(s):

Siva K. Panguluri ◽

Prasanna Kumar ◽

Subba R. Palli

Keyword(s):

Mammalian Cells ◽

Receptor Gene ◽

Functional Characterization ◽

Ecdysone Receptor ◽

Gene Switches

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A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport

Journal of Virology ◽

10.1128/jvi.79.8.5163-5173.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 5163-5173 ◽

Cited By ~ 81

Author(s):

Els Wessels ◽

Daniël Duijsings ◽

Richard A. Notebaart ◽

Willem J. G. Melchers ◽

Frank J. M. van Kuppeveld

Keyword(s):

Endoplasmic Reticulum ◽

Structural Model ◽

Ectopic Expression ◽

Rna Replication ◽

Secretory Protein ◽

Viral Rna ◽

Rich Region ◽

Golgi Transport ◽

Proline Rich Region

ABSTRACT The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

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