Protein quality control machinery supports primary ciliogenesis by eliminating GDP-bound Rab8-family GTPases.

The small GTPase Rab8 plays a vital role in the vesicular trafficking of cargo proteins from the trans-Golgi network to target membranes. Upon reaching its target destination, Rab8 is released from the vesicular membrane into the cytoplasm via guanosine triphosphate (GTP) hydrolysis. The fate of GDP-bound Rab8 released from the destination membranes, however, has not been investigated adequately. In this study, we found that GDP-bound Rab8 subfamily proteins are targeted for immediate degradation, and the pre-emptive quality control machinery is responsible for eliminating these proteins in a nucleotide-specific manner. We provide evidence that components of this quality control machinery have a critical role in vesicular trafficking events, including the formation of primary cilia, a process regulated by the Rab8 subfamily. These results suggest that the protein degradation machinery plays a critical role in the integrity of membrane trafficking by limiting the excessive accumulation of GDP-bound Rab8 subfamily proteins.

Leishmania major Strain-Dependent Macrophage Activation Contributes to Pathogenicity in the Absence of Lymphocytes.

Infection of C57BL/6 wild-type mice with Leishmania major 5-ASKH or Friedlin strains results in relatively similar pathogenicity with self-healing lesions within weeks. Parasite clearance depends on nitric oxide production by activated macrophages in response to cytokines produced mainly by CD4+ Th1 cells. In contrast, C57BL/6 Rag2 knockout mice, which lack T and B lymphocytes, show distinct pathologies during infection with these strains. Despite of the similar parasite number, the 5-ASKH infection induced severe inflammation rather than the Friedlin. To determine the immunological factors behind this phenomenon, we infected C57BL/6 Rag2 knockout mice with these two strains and compared immune cell kinetics and macrophage activation status. Compared with the Friedlin strain, the 5-ASKH strain elicited increased pathology associated with the accumulation of CD11bhigh, Ly6Ghigh neutrophils by week four and increased the expression of macrophage activation markers. We then analyzed the differentially expressed transcripts in infected bone marrow-derived macrophages by RNA sequencing. It showed upregulation of multiple inflammatory transcripts, including Toll-like receptor 1/2 (TLR1/2), CD69, and CARD14, upon 5-ASKH infection. Our findings suggest that different L. major strains can trigger distinct macrophage activation, contributing to the disease outcome observed in the absence of lymphocytes but not in the presence of lymphocytes. IMPORTANCE Disease manifestations of cutaneous leishmaniasis (CL) range from self-healing cutaneous lesions to chronic forms of the disease, depending on the infecting Leishmania sp. and host immune protection. Previous works on mouse models of CL show the distinct pathogenicity of Leishmania major strains in the absence of lymphocytes. However, the mechanisms of this pathology remain uncovered. In the trial to understand the immunological process involved in lymphocyte-independent pathology, we have found a specific induction of macrophages by different L. major strains that affect their ability to mount innate responses leading to neutrophilic pathology when lymphocytes are ablated.

BAG6 deficiency induces mis-distribution of mitochondrial clusters under depolarization.

Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin- and p62/sequestosome 1 (SQSTM1)-mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m-chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2-associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference-mediated depletion of endogenous BAG6 prevented Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/SQSTM1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.

Elimination of a signal sequence-uncleaved form of defective HLA protein through BAG6.

A portion of newly synthesized transmembrane domain proteins tend to fail to assemble correctly in the lumen of the endoplasmic reticulum, thus resulting in the production of a signal sequence-uncleaved form of the defective species. Although the efficient degradation of these mistargeted polypeptides is crucial, the molecular mechanism of their elimination pathway has not been adequately characterized. In this study, we focused on one such cryptic portion of a defective transmembrane domain protein, HLA-A, and show that a part of HLA-A is produced as a signal sequence-uncleaved labile species that is immediately targeted to the degradation pathway. We found that both BAG6 and proteasomes are indispensable for elimination of mislocalized HLA-A species. Furthermore, defective HLA-A is subjected to BAG6-dependent solubilization in the cytoplasm. These observations suggest that BAG6 acts as a critical factor for proteasome-mediated degradation of mislocalized HLA-A with a non-cleaved signal sequence at its N-terminus.

A conserved island of BAG6/Scythe is related to ubiquitin domains and participates in short hydrophobicity recognition.

BAG6 (also called Scythe) interacts with the exposed hydrophobic regions of newly synthesized proteins and escorts them to the degradation machinery through mechanisms that remain to be elucidated. In this study, we provide evidence that BAG6 physically interacts with the model defective protein substrate CL1 in a manner that depends directly on its short hydrophobicity. We found that the N terminus of BAG6 contains an evolutionarily conserved island tentatively designated the BAG6 ubiquitin-linked domain. Partial deletion of this domain in the BAG6 N-terminal fragment abolished in cell recognition of polyubiquitinated polypeptides as well as the hydrophobicity-mediated recognition of the CL1 degron in cell and in vitro. These observations suggest a mechanism whereby the BAG6 ubiquitin-linked domain provides a platform for discriminating substrates with shorter hydrophobicity stretches as a signal for defective proteins.