"Autophagy" is a highly conserved pathway for degradation, by which wasted intracellular macromolecules are delivered to lysosomes, where they are degraded into biologically active monomers such as amino acids that are subsequently re-used to maintain cellular metabolic turnover and homeostasis. Recent genetic studies have shown that mice lacking an autophagy-related gene (Atg5 or Atg7) cannot survive longer than 12 h after birth because of nutrient shortage. Moreover, tissue-speciWc impairment of autophagy in central nervous system tissue causes massive loss of neurons, resulting in neurodegeneration, while impaired autophagy in liver tissue causes accumulation of wasted organelles, leading to hepatomegaly. Although autophagy generally prevents cell death, our recent study using conditional Atg7-deWcient mice in CNS tissue has demonstrated the presence of autophagic neuron death in the hippocampus after neonatal hypoxic/ischemic brain injury. Thus, recent genetic studies have shown that autophagy is involved in various cellular functions. In this review, we introduce physiological and pathophysiological roles of autophagy.

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Exportin-5, an evolutionarily conserved nuclear export factor belonging to the importin-β family of proteins, is known to play a role in the nuclear export of small noncoding RNAs such as precursors of microRNA, viral minihelix RNA and a subset of tRNAs in mammalian cells. In this study, we show that the exportin-5 orthologues from different species such as human, fruit fly and yeast exhibit diverged functions. We found that Msn5p, a yeast exportin-5 orthologue, binds double-stranded RNAs and that it prefers a shorter 22 nt, double-stranded RNA to ∼80 nt pre-miRNA, even though both of these RNAs share a similar terminal structure. Furthermore, we found that Drosophila exportin-5 binds pre-miRNAs and that amongst the exportin-5 orthologues tested, it shows the highest affinity for tRNAs. The knockdown of Drosophila exportin-5 in cultured cells decreased the amounts of tRNA as well as miRNA, whereas the knock down of human exportin-5 in cultured cells affected only miRNA but not tRNA levels. These results indicate that double-stranded RNA binding ability is an inherited functional characteristic of the exportin-5 orthologues and that Drosophila exportin-5 functions as an exporter of tRNAs as well as pre-miRNAs in the fruit fly that lacks the orthologous gene for exportin-t.

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

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ATG9 is a membrane protein that is essential for autophagy and is considered to be directly involved in the early steps of autophagosome formation. Yeast Atg9 is mainly localized to small vesicles (Atg9 vesicles), whereas mammalian ATG9A is reportedly localized to the -Golgi network, the endosomal compartment, and other unidentified membrane structures. To dissect the ATG9A-containing membranes, we examined the subcellular localization of ATG9A and performed immunoisolation of those membranes. ATG9A-green fluorescent protein in human culture cells was observed as numerous puncta that move rapidly throughout the cytoplasm. We isolated these cytoplasmic membranes and found that they were small vesicles that resemble the yeast Atg9 vesicle. One of the proteins obtained proteomic analyses of the mammalian ATG9A vesicle was Rab1, a small GTPase that is essential in endoplasmic reticulum-to-Golgi vesicle trafficking. Knockdown studies of Rab1B showed a suppression of autophagy. In these Rab1B-depleted cells, ATG9A accumulated in intermediate membrane structures at autophagosome formation sites. These results indicate that Rab1B is involved in regulating the proper development of autophagosomes.-Kakuta, S., Yamaguchi, J., Suzuki, C., Sasaki, M., Kazuno, S., Uchiyama, Y. Small GTPase Rab1B is associated with ATG9A vesicles and regulates autophagosome formation.

(MShibata) S U M M A R Y Old and unneeded intracellular macromolecules are delivered through autophagy to lysosomes that degrade macromolecules into bioactive monomers such as amino acids. Autophagy is conserved in eukaryotes and is essential for the maintenance of cellular metabolism. Currently, more than 30 autophagy-related genes (Atgs) have been identified in yeast. Of these genes, the18 that are essential for autophagosome formation are also conserved in mammalian cells. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A protein (Atg9Ap) has been examined, little is known about its precise cell and tissue distribution. To determine this, we produced an antibody specific to mouse Atg9Ap. The antibody recognized both non-glycosylated and glycosylated Atg9Ap, which have molecular masses of ?94 kDa and 105 kDa, respectively. Although Atg9Ap was ubiquitously detected, it was highly expressed in neurons of the central nervous system. In Purkinje cells, Atg9Ap immunoreactivity was localized in the endoplasmic reticulum (ER), trans-Golgi network (TGN), lysosomes/late endosomes, and in axon terminals. These results suggest that Atg9Ap may be involved in autophagosome formation in the ER and axon terminals of neurons, the TGN, and lysosomes/late endosomes. (J Histochem Cytochem 58:443-453, 2010)