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Clathrin heavy chain 22 (CHC22) is an isoform of the well-characterized CHC17 clathrin heavy chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome-TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport. www.jcb.org/cgi/doi/10.1083/jcb.200908057

Intracellular trafficking of the glucose transporter GLUT4 from storage compartments to the plasma membrane is triggered in muscle and fat during the body's response to insulin. Clathrin is involved in intracellular trafficking, and in humans, the clathrin heavy-chain isoform CHC22 is highly expressed in skeletal muscle. We found a role for CHC22 in the formation of insulin-responsive GLUT4 compartments in human muscle and adipocytes. CHC22 also associated with expanded GLUT4 compartments in muscle from type 2 diabetic patients. Tissue-specific introduction of CHC22 in mice, which have only a pseudogene for this protein, caused aberrant localization of GLUT4 transport pathway components in their muscle, as well as features of diabetes. Thus, CHC22-dependent membrane trafficking constitutes a species-restricted pathway in human muscle and fat with potential implications for type 2 diabetes.

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells, esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

Byline: Chih-Ying Chen (1) Keywords: incentive compensation; investment opportunities; corporate governance; agency cost; director ownership Abstract: Governance scholars argue that outside directors have little incentive to monitor managers when their equity stake in the firm is not significant. A sample with a substantial level of outside director shareholdings is examined and a negative relationship between incentive compensation and outside director stock ownership is found. While firms pay higher incentive compensation when they have greater investment opportunities, the compensation contains excess pay due to ineffective corporate governance. Overall, the results suggest more effective corporate governance and lower incentive compensation when outside director stock ownership is higher. Author Affiliation: (1) Department of Accounting, School of Business and Management, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Article History: Registration Date: 13/10/2004

ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1(delta) synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2(delta) mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-(alpha)-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2(delta) cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1[Delta] allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1[Delta]). Most of the swa mutants exhibit phenotypes comparable to arf1[Delta] mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3[prime] end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 is mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.

Overexpression of the Schizosaccharomyces pombe byr4 gene suppresses cytokinesis and the need for the ras1 protein for sporulation. The initial steps of cytokinesis are normal but the later steps do not appear in the cells. The inhibition of cytokinesis is more severe in cells lacking ras1 and scd1. Cells with null byr4 mutations stop in late mitosis and undergo several cytokinesis and septation events. The karyokinesis in the mutant cells is defective as the two nuclei formed stain differently with 4',6-diamidino-2-phenylindole.

1. Introduction MicroRNAs (miRNAs) are small noncoding RNAs found in plants and animals which downregulate gene expression by binding to complementary sites on the 3' untranslated region (UTR) of target [...], MicroRNAs (miRNAs) downregulate gene expression by binding to the partially complementary sites in the 3' untranslated region (UTR) of target mRNAs. Several methods, such as Northern blot analysis, quantitative real-time RT-PCR, microarray, and the luciferase reporter system, are commonly used to quantify the relative level or activity of miRNAs. The disadvantage of these methods is the requirement for cell lysis, which means that several sets of wells/dishes of cells must be prepared to monitor changes in miRNA activity in time-course studies. In this study, we developed a multisampling reporter system in which two secretable bioluminescence-generating enzymes are employed, one as a reporter and the other as an internal control. The reporters consist of a pair of vectors containing the Metridia luciferase gene, one with and one without a duplicated miRNA targeting sequence at their 3'UTR, while the other vector coding for the secreted alkaline phosphatase gene is used as an internal control. This method allows miRNA activity to be monitored within the same population of cells over time by withdrawing aliquots of the culture medium. The practicability and benefits of this system are addressed in this report. Copyright [C] 2009 Pei-Chen Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.