The gene for Tom1 was initially identified as a specific target of the oncogene v-myb. The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain. VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated. In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains. Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex. In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif. Fluorescence microscopic analysis revealed that green fluorescent proteinTom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell. Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin.

ABSTRACT. RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.

The C/ebpb gene is translated into three different protein isoforms, two transcriptional activating proteins (38-kDa Full and 34-kDa liver-enriched transcriptional activation protein (LAP)) and one transcriptional inhibitory protein, by alternative use of different AUG initiation codons within the same open reading frame. The isoform 34-kDa LAP is thought to be the most transcriptionally active form of C/EBPβ in macrophages. To assess the function of the 34-kDa LAP in vivo, we generated knock-in mice, in which methionine 20 of C/EBPβ, the start site for the 34-kDa LAP is replaced with an alanine. The expression of the 34-kDa LAP was abolished in C/ebpbM20A/M20A mice. The induction of C/EBPβ target genes, such as inflammatory cytokines, chemokines, prostanoid synthetase, and antimicrobial peptides, was abolished in C/ebpbM20A/M20A macrophages, and C/ebpbM20A/M20A mice were susceptible to Listeria monocytogenes infection. Furthermore, the heat-killed Propionibacterium acnes-induced Th1 response, granuloma formation, and LPS shock were severely impaired. Nevertheless, impairment of intracellular bacteria killing, which is the most prominent phenotype in C/EBPβ-deficient mice, was not observed in C/ebpbM20A/M20A mice. Collectively, we demonstrated that 34-kDa LAP is responsible for NF-IL6-mediated gene induction, but not essential for intracellular bacteria killing in activated macrophages.

The Tom1 gene was initially identified as a specific target of retroviral oncogene v-Myb.1) This protein has an N-terminal VHS domain 2) followed by a short GAT domain.3) The crystal structures of the VHS domains of Tom1, 4) Hrs,5) and GGA proteins 6,7) have been determined. Although their global structures are similar, the VHS domains of GGA1 and GGA3, but not those of Tom1 and Hrs, have a specific recognition pocket for the mannose-6-phosphate receptor.6,7) Recently, the crystal structure of the GAT domain of GGA1 has been reported, [8][9][10] but its helical extension, which is indispensable for the association of GGA1 with ARF, is not conserved in the GAT domain of Tom1. Thus, the structurefunction relationships in the VHS and GAT domains of GGA proteins have been extensively elucidated, but those of Tom1 have not yet been determined. To investigate the function of Tom1, we previously carried out yeast two-hybrid screening using Tom1 as bait and found that Tollip is a Tom1-binding protein. 11)The adaptor protein Tollip was identified as an intermediate in IL-1-dependent signaling.12) IL-1 plays a central role in mediating a variety of inflammatory responses.13) The binding of IL-1 to the type I IL-1 receptor (IL-1RI) and the subsequent recruitment of the accessory protein IL-1RAcp result in their close spatial association, which allows the recruitment of the adaptor protein MyD88 and the IL-1R-associated-kinase (IRAK). In resting cells, Tollip forms a complex with IRAK and inhibits IL-1-induced signaling by blocking IRAK phosphorylation.12) In response to IL-1 signaling, IRAK becomes rapidly phosphorylated, dissociates from the receptor complex, and then associates with the downstream adaptor proteins such as TNF-associated factor 6 (TRAF6). The IL-1-induced signaling pathway bifurcates at TRAF6, 14,15) leading to the activation of the IkB kinase (IKK) complex and mitogen-activated protein kinases. These former and latter pathways are central to activation of the transcription factors nuclear factor (NF)-kB and AP-1, respectively. 16,17) In this study, we found that Tom1 inhibits the IL-1b-induced signaling pathway that leads to the activation of NFkB and AP-1, as Tollip does.12) On the other hand, in contrast to the action of Tollip, 12) Tom1 was found to inhibit the TNFa-induced signaling pathway, which also leads to the activation of NF-kB and AP-1. 18,19) To our knowledge, this is the first report of a common negative regulator of signaling pathways induced by IL-1b and TNF-a. MATERIALS AND METHODS Cell Culture and TransfectionHuman embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen). Transfection was performed using FuGene6 (Roche) according to the manufacturer's protocol.Plasmid Construction The open-reading frames of mouse Tom1, Tollip, and IL-1RI, all containing EcoRI and SalI sites, were amplified by PCR using the respective primers. The PCR fragments were subcloned into the pGE...