Monthly air temperature in Hiroshima, Japan from January 2009 to December 2013.

<p>Mean monthly air temperature (closed circles), maximum air temperature (closed squares), and minimum air temperature (open squares) in South Ward of Hiroshima City. Data obtained from the Japan Meteorological Agency.</p

Seasonal variation in the number of patients initiating dialysis.

<p>(A) Number of all patients initiating dialysis. (B) Number initiating dialysis according to age. (C) Number initiating dialysis among patients aged <65 years (Group A) (left) and ≥65 years (Group B) (right).</p

Clinical characteristics.

<p>Clinical characteristics.</p

Seasonal variations in conditions at dialysis initiation.

<p>Seasonal variations in conditions at dialysis initiation.</p

Seasonal variation in number of patients according to main symptoms at dialysis initiation.

<p>Number of patients with fluid overload, general malaise, nausea or appetite loss, hyperkalemia, other uremic symptoms, and no symptom at dialysis initiation among Groups A and B.</p

Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183

<div><p>We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases.</p></div

Proteomics analysis of the RNF183 interacting protein.

<p>Proteomics analysis of the RNF183 interacting protein.</p

Effects of Sec16 on RNF183 protein stability.

<p>(A) Effect of Sec16A downregulation on RNF183 protein stability. HeLa cells stably expressing RNF183-V5 were transfected with NC (1st, 3rd, and 5th panels) or Sec16A (2nd, 4th and 6th panels) siRNA. At 44 h after transfection, cells were treated with 30 μg/ml cycloheximide (CHX) and 10 μM MG132 for the indicated periods. Total cell lysates were analyzed by Western blotting with an anti-V5 (1st and 2nd panels), Sec16A (3rd and 4th panels), and β-actin (5th and 6th panels) antibodies. (B) Quantitative curves of data from (A). RNF183 levels at each time point were plotted relative to the level at time 0 (n = 3). Asterisks represent significant differences (Student’s t test with Bonferroni correction, *p < 0.05; NC vs. Sec16A siRNA; #p < 0.05, ##p < 0.01; ##p < 0.001; Sec16A siRNA vs. Sec16A siRNA + MG132).</p

Effects of Sec16 on other ubiquitin ligases.

<p>(A) Interactions of RNF152 and HRD1 with Sec16A. Coimmunoprecipitation was performed in HEK293 cells engineered to stably express V5-tagged RNF183, V5-tagged RNF152, or myc-tagged HRD1. Cell lysates were immunoprecipitated with anti-V5 or anti-myc antibodies or normal mouse immunoglobulin G (IgG; negative control). Immune complexes were analyzed by Western blotting with an anti-Sec16A antibody (<i>top</i> panel) and anti-V5 (<i>second</i> panel) or anti-myc antibodies (<i>third</i> panel). (B, D) Effect of Sec16A downregulation on RNF152 and HRD1 protein stability. Stable RNF152-V5- or HRD1-myc-expressing HEK293 cells were transfected with NC or Sec16A siRNA. At 48 h after transfection, cells were subjected to a CHX assay. (C, E) Asterisks represent significant differences (n = 3; Student’s t test with Bonferroni correction, *p < 0.05, ***p < 0.001; NC vs. Sec16A siRNA).</p

Interaction and colocalization of RNF183 and Sec16A.

<p>(A) Interaction of RNF183 and Sec16A. Cell lysates from HEK293 cells stably expressing V5-tagged RNF183 were immunoprecipitated using anti-V5 antibody, and the immune complexes were analyzed by Western blotting with anti-Sec16A (<i>upper</i> panel) or anti-V5 (<i>lower</i> panel) antibodies. (B) Colocalization of RNF183 with Sec16A. HeLa cells stably transfected with RNF183-V5 were subjected to immunofluorescence staining with anti-Sec16A (<i>green</i>) and DAPI (<i>blue</i>). (C) Effect of RNF183 knockdown on Sec16A protein. RNF183 expression in HEK293 cells stably transfected with RNF183-V5 was suppressed using siRNA. Endogenous Sec16A protein levels were detected using Western blotting with anti-Sec16A antibody. NC, negative control. (D) Schematic diagram of the predicted domains of Sec16A. (E) Interaction domain of Sec16A with RNF183. Lysates from HEK293 cells stably expressing RNF183 transiently transfected with GFP-tagged full-length Sec16A (Full) or its deletion mutant constructs were subjected to immunoprecipitation with anti-V5 antibody, followed by immunoblotting with anti-GFP antibody.</p