scholarly journals COMMD1 and PtdIns(4,5)P2 interaction maintain ATP7B copper transporter trafficking fidelity in HepG2 cells

2019 ◽  
Author(s):  
Davis J. Stewart ◽  
Kristopher K. Short ◽  
Breanna N. Maniaci ◽  
Jason L. Burkhead
Keyword(s):  
Protein Trafficking ◽  
Early Endosome ◽  
Copper Accumulation ◽  
Direct Function ◽  
Copper Transporter ◽  
Membrane Protein Trafficking ◽  
Copper Balance ◽  
Cellular Copper ◽  
Golgi Network ◽  
Specific Membrane

ABSTRACTCopper-responsive intracellular ATP7B trafficking is critical to maintain copper balance in mammalian hepatocytes and thus organismal copper levels. The COMMD1 protein binds both the ATP7B copper transporter and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2), while COMMD1 loss causes hepatocyte copper accumulation. Although it is clear that COMMD1 is included in endocytic trafficking complexes, a direct function for COMMD1 in ATP7B trafficking has not been defined. In this study, experiments using quantitative reveal that COMMD1 modulates the copper-responsive ATP7B trafficking through recruitment to PtdIns(4,5)P2. Decreased COMMD1 abundance results in loss of ATP7B from lysosomes and thetrans-Golgi network (TGN) in high copper conditions, while excess expression of COMMD1 also disrupts ATP7B trafficking and TGN structure. Overexpression of COMMD1 mutated to inhibit PtdIns(4,5)P2binding has little impact on ATP7B trafficking. A mechanistic PtdIns(4,5)P2-mediated function for COMMD1 is proposed that is consistent with decreased cellular copper export due to disruption of the ATP7B trafficking itinerary and accumulation in the early endosome when COMMD1 is depleted. PtdIns(4,5)P2interaction with COMMD1 as well as COMMD1 abundance may both be important in maintenance of specific membrane protein trafficking pathways.SUMMARYQuantitative analysis of 3D protein colocalization defines the cellular function of COMMD1 in maintenance of ATP7B copper transporter trafficking fidelity and the importance of PtdIns(4,5)P2in this action.

scholarly journals Functional Characterization of CgCTR2, a Putative Vacuole Copper Transporter That Is Involved in Germination and Pathogenicity in Colletotrichum gloeosporioides

2008 ◽  
Vol 7 (7) ◽  
pp. 1098-1108 ◽  
Author(s):  
Sima Barhoom ◽  
Martin Kupiec ◽  
Xinhua Zhao ◽  
Jin-Rong Xu ◽  
Amir Sharon
Keyword(s):  
Colletotrichum Gloeosporioides ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Sod Activity ◽  
Copper Transporter ◽  
Putative Gene ◽  
Copper Balance ◽  
Increased Sensitivity ◽  
Green Fluorescent ◽  
Cellular Copper

ABSTRACT Copper is a cofactor and transition metal involved in redox reactions that are essential in all eukaryotes. Here, we report that a vacuolar copper transporter that is highly expressed in resting spores is involved in germination and pathogenicity in the plant pathogen Colletotrichum gloeosporioides. A screen of C. gloeosporioides transformants obtained by means of a promoterless green fluorescent protein (GFP) construct led to the identification of transformant N159 in which GFP signal was observed in spores. The transforming vector was inserted 70 bp upstream of a putative gene with homology to the Saccharomyces cerevisiae vacuolar copper transporter gene CTR2. The C. gloeosporioides CTR2 (CgCTR2) gene fully complemented growth defects of yeast ctr2Δ mutants, and a CgCTR2-cyan fluorescent protein (CFP) fusion protein accumulated in vacuole membranes, confirming the function of the protein as a vacuolar copper transporter. Expression analysis indicated that CgCTR2 transcript is abundant in resting conidia and during germination in rich medium and downregulated during “pathogenic” germination and the early stages of plant infection. CgCTR2 overexpression and silencing mutants were generated and characterized. The Cgctr2 mutants had markedly reduced Cu superoxide dismutase (SOD) activity, suggesting that CgCTR2 is important in providing copper to copper-dependent cytosolic activities. The Cgctr2-silenced mutants had increased sensitivity to H2O2 and reduced germination rates. The mutants were also less virulent to plants, but they did not display any defects in appressorium formation and penetration efficiency. An external copper supply compensated for the hypersensitivity to H2O2 but not for the germination and pathogenicity defects of the mutants. Similarly, overexpression of CgCTR2 enhanced resistance to H2O2 but had no effect on germination or pathogenicity. Our results show that copper is necessary for optimal germination and pathogenicity and that CgCTR2 is involved in regulating cellular copper balance during these processes.

scholarly journals Retromer retrieves the Wilson Disease protein, ATP7B from lysosomes in a copper-dependent mode

2020 ◽  
Author(s):  
Santanu Das ◽  
Saptarshi Maji ◽  
Ruturaj ◽  
Indira Bhattacharya ◽  
Tanusree Saha ◽  
...  
Keyword(s):  
Super Resolution ◽  
Copper Level ◽  
Copper Transporter ◽  
Copper Chelation ◽  
Proximity Ligation ◽  
High Copper ◽  
Core Member ◽  
Disease Protein ◽  
Cellular Copper ◽  
Golgi Network

ATP7B utilizes lysosomal exocytosis to export copper from hepatocytes. We investigated the fate of ATP7B, post-copper export. At high copper ATP7B traffics to lysosomes and upon subsequent copper chelation, returns to Trans Golgi Network. At high copper, ATP7B co-localizes with lysosomal marker, Lamp1 and the core member of retromer complex, Vps35. Knocking down VPS35 did not alter copper-responsive vesicularization of ATP7B; rather upon subsequent copper chelation, ATP7B failed to relocalize to TGN that could be rescued by overexpressing wtVPS35. Using super-resolution microscopy and proximity ligation assays we demonstrate that VPS35 and ATP7B are juxtaposed on the same lysosomal compartment and their interaction is indirect. Utilizing in-cell photoamino acid-based UV-crosslinking and subsequent immunoprecipitation, we detected ATP7B and retromer subunits, VPS35 and VPS26 in a large complex in high copper conditions, hence confirming their interaction. We demonstrate that retromer regulates lysosome to TGN trafficking of the copper transporter ATP7B and it is dependent upon cellular copper level.

Roles of COMM-domain-containing 1 in stability and recruitment of the copper-transporting ATPase in a mouse hepatoma cell line

2010 ◽  
Vol 429 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Takamitsu Miyayama ◽  
Daisuke Hiraoka ◽  
Fumika Kawaji ◽  
Emi Nakamura ◽  
Noriyuki Suzuki ◽  
...  
Keyword(s):  
Cell Line ◽  
Hepatoma Cell ◽  
Copper Accumulation ◽  
Copper Level ◽  
Hepatoma Cell Line ◽  
Copper Transporter ◽  
Trans Golgi Network ◽  
Mouse Hepatoma ◽  
Cytoplasmic Vesicles ◽  
Golgi Network

A novel function of COMMD1 {COMM [copper metabolism MURR1 (mouse U2af1-rs1 region 1)]-domain-containing 1}, a protein relevant to canine copper toxicosis, was examined in the mouse hepatoma cell line Hepa 1-6 with multi-disciplinary techniques consisting of molecular and cellular biological techniques, speciation and elemental imaging. To clarify the function of COMMD1, COMMD1-knockdown was accomplished by introducing siRNA (small interfering RNA) into the cells. Although COMMD1-knockdown did not affect copper incorporation, it inhibited copper excretion, resulting in copper accumulation, which predominantly existed in the form bound to MT (metallothionein). It is known that the liver copper transporter Atp7b (ATP-dependent copper transporter 7β), localizes on the trans-Golgi network membrane under basal copper conditions and translocates to cytoplasmic vesicles to excrete copper when its concentration exceeds a certain threshold, with the vesicles dispersing in the periphery of the cell. COMMD1-knockdown reduced the expression of Atp7b, and abolished the relocation of Atp7b back from the periphery to the trans-Golgi network membrane when the copper concentration was reduced by treatment with a Cu(I) chelator. The same phenomena were observed during COMMD1-knockdown when another Atp7b substrate, cis-diamminedichloroplatinum, and its sequestrator, glutathione ethylester, were applied. These results suggest that COMMD1 maintains the amount of Atp7b and facilitates recruitment of Atp7b from cytoplasmic vesicles to the trans-Golgi network membrane, i.e. COMMD1 is required to shuttle Atp7b when the intracellular copper level returns below the threshold.

scholarly journals BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis

2012 ◽  
Vol 24 (7) ◽  
pp. 3074-3086 ◽  
Author(s):  
Elena Feraru ◽  
Mugurel I. Feraru ◽  
Rin Asaoka ◽  
Tomasz Paciorek ◽  
Riet De Rycke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Protein Trafficking ◽  
Plasma Membrane Protein ◽  
Trans Golgi Network ◽  
Membrane Protein Trafficking ◽  
Golgi Network

scholarly journals A rapid and affordable screening platform for membrane protein trafficking

BMC Biology ◽  
2015 ◽  
Vol 13 (1) ◽  
Author(s):  
Joshua C. Snyder ◽  
Thomas F. Pack ◽  
Lauren K. Rochelle ◽  
Subhasish K. Chakraborty ◽  
Ming Zhang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Membrane Protein Trafficking ◽  
Screening Platform

Ethanol and Membrane Protein Trafficking: Diverse Mechanisms of Ethanol Action

2002 ◽  
Vol 26 (2) ◽  
pp. 287-293 ◽  
Author(s):  
Laura E. Nagy ◽  
M. Raj Lakshman ◽  
Carol A. Casey ◽  
Cynthia F. Bearer
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Membrane Protein Trafficking

scholarly journals Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane

2012 ◽  
Vol 23 (15) ◽  
pp. 2917-2929 ◽  
Author(s):  
Emily Deutsch ◽  
Aubrey V. Weigel ◽  
Elizabeth J. Akin ◽  
Phil Fox ◽  
Gentry Hansen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Ion Channel ◽  
Protein Trafficking ◽  
Hippocampal Neurons ◽  
Surface Membrane ◽  
Cell Types ◽  
Homogeneous Surface ◽  
Hek Cells ◽  
Membrane Protein Trafficking

Voltage-gated K+ (Kv) channels regulate membrane potential in many cell types. Although the channel surface density and location must be well controlled, little is known about Kv channel delivery and retrieval on the cell surface. The Kv2.1 channel localizes to micron-sized clusters in neurons and transfected human embryonic kidney (HEK) cells, where it is nonconducting. Because Kv2.1 is postulated to be involved in soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion, we examined the hypothesis that these surface clusters are specialized platforms involved in membrane protein trafficking. Total internal reflection–based fluorescence recovery after photobleaching studies and quantum dot imaging of single Kv2.1 channels revealed that Kv2.1-containing vesicles deliver cargo at the Kv2.1 surface clusters in both transfected HEK cells and hippocampal neurons. More than 85% of cytoplasmic and recycling Kv2.1 channels was delivered to the cell surface at the cluster perimeter in both cell types. At least 85% of recycling Kv1.4, which, unlike Kv2.1, has a homogeneous surface distribution, is also delivered here. Actin depolymerization resulted in Kv2.1 exocytosis at cluster-free surface membrane. These results indicate that one nonconducting function of Kv2.1 is to form microdomains involved in membrane protein trafficking. This study is the first to identify stable cell surface platforms involved in ion channel trafficking.

scholarly journals In vivo reconstitution finds multivalent RNA-RNA interactions as drivers of mesh-like condensates

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Weirui Ma ◽  
Gang Zheng ◽  
Wei Xie ◽  
Christine Mayr
Keyword(s):  
Surface Area ◽  
Protein Trafficking ◽  
Rna Binding ◽  
Interaction Network ◽  
Large Surface Area ◽  
High Propensity ◽  
Membrane Protein Trafficking ◽  
Protein Components

Liquid-like condensates have been thought to be sphere-like. Recently, various condensates with filamentous morphology have been observed in cells. One such condensate is the TIS granule network that shares a large surface area with the rough endoplasmic reticulum and is important for membrane protein trafficking. It has been unclear how condensates with mesh-like shapes, but dynamic protein components are formed. In vitro and in vivo reconstitution experiments revealed that the minimal components are a multivalent RNA-binding protein that concentrates RNAs that are able to form extensive intermolecular mRNA-mRNA interactions. mRNAs with large unstructured regions have a high propensity to form a pervasive intermolecular interaction network that acts as condensate skeleton. The underlying RNA matrix prevents full fusion of spherical liquid-like condensates, thus driving the formation of irregularly shaped membraneless organelles. The resulting large surface area may promote interactions at the condensate surface and at the interface with other organelles.

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