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.

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.

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.