Decision letter: A Ras-like domain in the light intermediate chain bridges the dynein motor to a cargo-binding region

Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). In this study, we have determined the crystal structure of the conserved LIC domain, which binds the motor heavy chain, from a thermophilic fungus. We show that the LIC has a Ras-like fold with insertions that distinguish it from Ras and other previously described G proteins. Despite having a G protein fold, the fungal LIC has lost its ability to bind nucleotide, while the human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport. These studies provide the first structural information and insight into the evolutionary origin of the LIC as well as revealing how this critical subunit connects the dynein motor to cargo.

Download Full-text

Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis in Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.12.10.2921 ◽

2001 ◽

Vol 12 (10) ◽

pp. 2921-2933 ◽

Cited By ~ 48

Author(s):

John H. Yoder ◽

Min Han

Keyword(s):

Caenorhabditis Elegans ◽

Nucleotide Binding ◽

Cytoplasmic Dynein ◽

Meiotic Spindle ◽

Phenotypic Characterization ◽

Loss Of Function ◽

Dynein Motor ◽

Centrosome Separation ◽

Transporter Family ◽

Intermediate Chain

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations indli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of thedli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-celldli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective indli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1mutant background, indicating this is not an essential domain for DLI-1 function.

Download Full-text

DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor

FEBS Journal ◽

10.1111/febs.13388 ◽

2015 ◽

Vol 282 (20) ◽

pp. 3945-3958 ◽

Cited By ~ 8

Author(s):

Javier Merino-Gracia ◽

María Flor García-Mayoral ◽

Peter Rapali ◽

Ruth Ana Valero ◽

Marta Bruix ◽

...

Keyword(s):

Small Gtpase ◽

Dynein Motor ◽

Dynein Intermediate Chain ◽

Intermediate Chain

Download Full-text

The Gene for the Intermediate Chain Subunit of Cytoplasmic Dynein Is Essential in Drosophila

Genetics ◽

10.1093/genetics/162.3.1211 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1211-1220 ◽

Cited By ~ 2

Author(s):

Kristin L M Boylan ◽

Thomas S Hays

Keyword(s):

Intracellular Transport ◽

De Novo ◽

Cytoplasmic Dynein ◽

Chain Gene ◽

Temperature Sensitive ◽

Wing Development ◽

Dynein Motor ◽

Dynein Intermediate Chain ◽

Microtubule Motor ◽

Intermediate Chain

Abstract The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued1, a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.

Download Full-text

Ferritin, an Iron Storage Protein, Associates with Kinesin 1 through the Cargo-binding Region of Kinesin Heavy Chains (KHCs)

Journal of Life Science ◽

10.5352/jls.2016.26.6.698 ◽

2016 ◽

Vol 26 (6) ◽

pp. 698-704 ◽

Cited By ~ 1

Author(s):

Won Hee Jang ◽

Young Joo Jeong ◽

Won Hee Lee ◽

Mooseong Kim ◽

Sang-Jin Kim ◽

...

Keyword(s):

Storage Protein ◽

Binding Region ◽

Iron Storage ◽

Heavy Chains ◽

Iron Storage Protein ◽

Cargo Binding

Download Full-text

Cellular and Viral Determinants of HSV-1 Entry and Intracellular Transport towards Nucleus of Infected Cells

Journal of Virology ◽

10.1128/jvi.02434-20 ◽

2021 ◽

Author(s):

Farhana Musarrat ◽

Vladimir Chouljenko ◽

Konstantin G. Kousoulas

Keyword(s):

Post Infection ◽

Intracellular Transport ◽

Virus Entry ◽

Motor Complex ◽

Kinase C ◽

Dynein Motor ◽

Dynein Intermediate Chain ◽

Infected Cells ◽

Hsv 1 ◽

Intermediate Chain

HSV-1 employs cellular motor proteins and modulates kinase pathways to facilitate intracellular virion capsid transport. Previously, we and others have shown that the Akt inhibitor miltefosine inhibited virus entry. Herein, we show that the protein kinase C inhibitors staurosporine (STS) and gouml inhibited HSV-1 entry into Vero cells, and that miltefosine prevents HSV-1 capsid transport toward the nucleus. We have reported that the HSV-1 UL37 tegument protein interacts with the dynein motor complex during virus entry and virion egress, while others have shown that the UL37/UL36 protein complex binds dynein and kinesin causing a saltatory movement of capsids in neuronal axons. Co-immoprecipitation experiments confirmed previous findings from our laboratory that the UL37 protein interacted with the dynein intermediate chain (DIC) at early times post infection. This UL37-DIC interaction was concurrent with DIC phosphorylation in infected, but not mock-infected cells. Miltefosine inhibited dynein phosphorylation when added before, but not after virus entry. Inhibition of motor accessory protein dynactins (DCTN2, DCTN3), the adaptor proteins EB1 and the Bicaudal D homolog 2 (BICD2) expression using lentiviruses expressing specific shRNAs, inhibited intracellular transport of virion capsids toward the nucleus of human neuroblastoma (SK-N-SH) cells. Co-immunoprecipitation experiments revealed that the major capsid protein Vp5 interacted with dynactins (DCTN1/p150 and DCTN4/p62) and the end-binding protein (EB1) at early times post infection. These results show that Akt and kinase C are involved in virus entry and intracellular transport of virion capsids, but not in dynein activation via phosphorylation. Importantly, both the UL37 and Vp5 viral proteins are involved in dynein-dependent transport of virion capsids to the nuclei of infected cells. Importance. Herpes simplex virus type-1 enter either via fusion at the plasma membranes or endocytosis depositing the virion capsids into the cytoplasm of infected cells. The viral capsids utilize the dynein motor complex to move toward the nuclei of infected cells using the microtubular network. This work shows that inhibitors of the Akt kinase and kinase C inhibit not only viral entry into cells but also virion capsid transport toward the nucleus. In addition, the work reveals that the virion protein ICP5 (VP5) interacts with the dynein cofactor dynactin, while the UL37 protein interacts with the dynein intermediate chain (DIC). Importantly, neither Akt nor Kinase C was found to be responsible for phosphorylation/activation of dynein indicating that other cellular or viral kinases may be involved.

Download Full-text

Cellular and Viral Determinants of HSV-1 Entry and Intracellular Transport towards Nucleus of Infected Cells

10.1101/2021.01.04.425350 ◽

2021 ◽

Author(s):

Farhana Musarrat ◽

Vladimir Chouljenko ◽

Konstantin G. Kousoulas

Keyword(s):

Post Infection ◽

Intracellular Transport ◽

Virus Entry ◽

Motor Complex ◽

Kinase C ◽

Dynein Motor ◽

Dynein Intermediate Chain ◽

Infected Cells ◽

Hsv 1 ◽

Intermediate Chain

AbstractHSV-1 employs cellular motor proteins and modulates kinase pathways to facilitate intracellular virion capsid transport. Previously, we and others have shown that the Akt inhibitor miltefosine inhibited virus entry. Herein, we show that the protein kinase C inhibitors staurosporine (STS) and gouml inhibited HSV-1 entry into Vero cells, and that miltefosine prevents HSV-1 capsid transport toward the nucleus. We have reported that the HSV-1 UL37 tegument protein interacts with the dynein motor complex during virus entry and virion egress, while others have shown that the UL37/UL36 protein complex binds dynein and kinesin causing a saltatory movement of capsids in neuronal axons. Co-immoprecipitation experiments confirmed previous findings from our laboratory that the UL37 protein interacted with the dynein intermediate chain (DIC) at early times post infection. This UL37-DIC interaction was concurrent with DIC phosphorylation in infected, but not mock-infected cells. Miltefosine inhibited dynein phosphorylation when added before, but not after virus entry. Inhibition of motor accessory protein dynactins (DCTN2, DCTN3), the adaptor proteins EB1 and the Bicaudal D homolog 2 (BICD2) expression using lentiviruses expressing specific shRNAs, inhibited intracellular transport of virion capsids toward the nucleus of human neuroblastoma (SK-N-SH) cells. Co-immunoprecipitation experiments revealed that the major capsid protein Vp5 interacted with dynactins (DCTN1/p150 and DCTN4/p62) and the end-binding protein (EB1) at early times post infection. These results show that Akt and kinase C are involved in virus entry and intracellular transport of virion capsids, but not in dynein activation via phosphorylation. Importantly, both the UL37 and Vp5 viral proteins are involved in dynein-dependent transport of virion capsids to the nuclei of infected cells.ImportanceHerpes simplex virus type-1 enter either via fusion at the plasma membranes or endocytosis depositing the virion capsids into the cytoplasm of infected cells. The viral capsids utilize the dynein motor complex to move toward the nuclei of infected cells using the microtubular network. This work shows that inhibitors of the Akt kinase and kinase C inhibit not only viral entry into cells but also virion capsid transport toward the nucleus. In addition, the work reveals that the virion protein ICP5 (VP5) interacts with the dynein cofactor dynactin, while the UL37 protein interacts with the dynein intermediate chain (DIC). Importantly, neither Akt nor Kinase C was found to be responsible for phosphorylation/activation of dynein indicating that other cellular or viral kinases may be involved.

Download Full-text

Interactions of LC8 with N-Terminal Segments of the Intermediate Chain of Cytoplasmic Dynein

The Scientific World JOURNAL ◽

10.1100/tsw.2003.56 ◽

2003 ◽

Vol 3 ◽

pp. 647-654 ◽

Cited By ~ 2

Author(s):

Afua Nyarko ◽

Michael Hare ◽

Moses Makokha ◽

Elisar Barbar

Keyword(s):

Limited Proteolysis ◽

Coiled Coil ◽

Cytoplasmic Dynein ◽

Light Chains ◽

Ordered Structure ◽

Dynein Motor ◽

Dynein Light Chains ◽

Significant Conformational Change ◽

Intermediate Chain

LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8.

Download Full-text