The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cationindependent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.

Cathepsin D-deficient (CDϪ/Ϫ) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/ autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CDϪ/Ϫ mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CDϪ/Ϫ mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CDϪ/Ϫ neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CDϪ/Ϫ mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region. We found that overproduction of Apg14p partially reversed the defect in autophagy induced by the apg6-1 mutation. The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole. However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-1 mutant, nor did the apg14 null mutation affect the CPY sorting pathway. Structural analysis of APG6 revealed that APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late- 137, 79 -92). Subcellular fractionation indicated thatApg14p and Apg6p peripherally associated with a membrane structure(s). Apg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex. These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway. Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway.

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Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.