Microbial degradation of clothianidin was characterized under aerobic and anaerobic California rice field conditions. Rate constants (k) and half-lives (DT50) were determined for aerobic and anaerobic microcosms, and an enrichment experiment was performed at various nutrient conditions and pesticide concentrations. Temperature effects on anaerobic degradation rates were determined at 22 ± 2 and 35 ± 2 °C. Microbial growth was assessed in the presence of various pesticide concentrations, and distinct colonies were isolated and identified. Slow aerobic degradation was observed, but anaerobic degradation occurred rapidly at both 25 and 35 °C. Transformation rates and DT50 values in flooded soil at 35 ± 2 °C (k = -7.16 × 10(-2) ± 3.08 × 10(-3) day(-1), DT50 = 9.7 days) were significantly faster than in 25 ± 2 °C microcosms (k= -2.45 × 10(-2) ± 1.59 × 10(-3) day(-1), DT50 = 28.3 days). At the field scale, biodegradation of clothianidin will vary with extent of oxygenation.

Copper-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 localized to endocytic trafficking complexes, a direct function for COMMD1 in ATP7B trafficking is not defined. In this study, experiments using quantitative colocalization analysis 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 the trans-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)P2 binding 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 early endosome accumulation when COMMD1 is depleted. PtdIns(4,5)P2 interaction with COMMD1 as well as COMMD1 abundance may both be important in maintenance of specific membrane protein trafficking pathways.

8Copper-responsive intracellular ATP7B trafficking is critical to maintain copper balance 9 in mammalian hepatocytes and thus organismal copper levels. The COMMD1 protein binds 10 both the ATP7B copper transporter and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2), 11 while COMMD1 loss causes hepatocyte copper accumulation. Although it is clear that 12 COMMD1 is included in endocytic trafficking complexes, a direct function for COMMD1 in 13 ATP7B trafficking has not been defined. In this study, experiments using quantitative reveal that 14 COMMD1 modulates the copper-responsive ATP7B trafficking through recruitment to 15 PtdIns(4,5)P2. Decreased COMMD1 abundance results in loss of ATP7B from lysosomes and 16 the trans-Golgi network (TGN) in high copper conditions, while excess expression of COMMD1 17 also disrupts ATP7B trafficking and TGN structure. Overexpression of COMMD1 mutated to 18 inhibit PtdIns(4,5)P2 binding has little impact on ATP7B trafficking. A mechanistic PtdIns(4,5)P2-19 mediated function for COMMD1 is proposed that is consistent with decreased cellular copper 20 export due to disruption of the ATP7B trafficking itinerary and accumulation in the early 21 endosome when COMMD1 is depleted. PtdIns(4,5)P2 interaction with COMMD1 as well as 22 COMMD1 abundance may both be important in maintenance of specific membrane protein 23 trafficking pathways. 24 25 SUMMARY 26Quantitative analysis of 3D protein colocalization defines the cellular function of COMMD1 in 27 maintenance of ATP7B copper transporter trafficking fidelity and the importance of PtdIns(4,5)P2 28 in this action. 29

Copper is essential for all life due to its role as a catalyst in redox reactions. However, it must be tightly regulated as excess copper can cause oxidative damage to the cell. One example of copper dyshomeostasis is the autosomal recessive condition is known as Wilson disease which can result in liver disease and neurological problems. To maintain copper homeostasis, cells rely on a system of chaperone proteins as well as three copper transporters; CRT1, ATP7A, and ATP7B. ATP7B is a large multidomain membrane‐spanning protein that exports copper from the cytosol and is highly expressed in liver, brain, and kidney cells. ATP7B has a dual role in maintaining copper homeostasis. In the liver, ATP7B functions in the trans‐Golgi network where it pumps copper into the lumen for biosynthesis of secreted cuproproteins. When cellular copper levels are elevated, ATP7B traffics in vesicles towards the apical membrane to move excess copper into bile for excretion from the body. This proves to be a complex decision for the cell as it must retain some ATP7B at the trans‐Golgi for continued biosynthesis of cuproproteins while simultaneously removing the excess. When copper levels are low, ATP7B returns to the Golgi, though it is likely that some is degraded. Although maintaining proper copper homeostasis is important, the mechanism that influences ATP7B distribution is poorly understood. Since being identified as mutated in Bedlington Terriers exhibiting canine copper toxicosis, the protein COMMD1 has been of particular interest as a regulator of cellular copper levels. COMMD1 exhibits both Phosphatidylinositol (PdtIns) effector and Ubiquitin ligase adaptor activity. COMMD1 interacts with a number of membrane proteins including ATP7B. Importantly, COMMD1 downregulation affects cellular copper levels. To investigate the relationship between COMMD1 and ATP7B we used quantitative immunofluorescence microscopy in HepG2 hepatoma cells to analyze the subcellular locations of ATP7B in response to misexpression of COMMD1. The overexpression of the COMMD1 T174M mutant, which was first identified in an atypical Wilson disease patient, significantly decreased the Golgi‐associated fraction of ATP7B and colocalization between ATP7B and lysosomes was also reduced during retrograde trafficking. The T174M mutation may affect PtdIns(4,5)P2 binding by COMMD1, thus we believe that PtdIns(4,5)P2 recruits and facilitates a functional interaction between COMMD1 and ATP7B.Support or Funding InformationNational Science Foundation (MCB‐1411890), NIH 8P20GM103395‐12 (JLB Sub‐Project)