We found that the autophagic machinery could effectively eliminate pathogenic group A Streptococcus (GAS) within nonphagocytic cells. After escaping from endosomes into the cytoplasm, GAS became enveloped by autophagosome-like compartments and were killed upon fusion of these compartments with lysosomes. In autophagy-deficient Atg5-/- cells, GAS survived, multiplied, and were released from the cells. Thus, the autophagic machinery can act as an innate defense system against invading pathogens.

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca2؉ -binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human Alix⌬C (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports. When a mutant of SKD1 that lacks ATPase activity [SKD1(E235Q)] was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments). Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments. In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments. Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D. An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes). Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes. We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5 −/− cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5 −/− cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

ABSTRACT. Mouse SKD1 AAA ATPase is involved in the sorting and transport from endosomes; cells overexpressing a dominant-negative mutant, SKD1 E235Q were defective in endosomal transport to both the plasma membranes and lysosomes . In the present study, we demonstrated that overexpression of SKD1 E235Q using an adenovirus delivery system caused a defect in autophagy-dependent bulk protein degradation. Morphological observations suggested that this inhibition of autophagy results from an impairment of autolysosome formation. SKD1 E235Q overexpression also inhibited transport from endosomes to autophagosomes, an event normally occurring prior to fusion with lysosomes. These results indicate that SKD1-dependent endosomal membrane trafficking is required for formation of autolysosomes.Key words: endocytosis/autophagy/membrane traffic/adenovirus-mediated expression system Cells take up extracellular macromolecules into early endosomes by endocytosis. The cargo is then either sorted to late endosome-lysosomes or recycled back to the plasma membrane (Corvera et al

Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments. In this paper, we demonstrate that leucine-rich repeat kinase LRRK1, which contains a MAPKKK-like kinase domain, forms a complex with activated EGFR through an interaction with Grb2. Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes. LRRK1 regulates EGFR transport from early to late endosomes and regulates the motility of EGF-containing early endosomes in a manner dependent on its kinase activity. Furthermore, LRRK1 serves as a scaffold facilitating the interaction of EGFR with the endosomal sorting complex required for transport-0 complex, thus enabling efficient sorting of EGFR to the inner vesicles of multivesicular bodies. Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.

Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.