As one of the major degradative mechanisms conserved among eukaryotic cells, autophagy mediates the turnover and recycling of long-lived cytosolic proteins and excess or damaged organelles (Klionsky, 2005
). The cargo destined for autophagic degradation is sequestered in a double-membrane vesicle called an autophagosome, which fuses with the lysosome in mammalian cells or the vacuole in yeast. Eventually, the cargo is degraded by lysosomal/vacuolar resident hydrolases. Autophagy occurs in response to physiological stress or developmental signals (Levine and Klionsky, 2004
). Recently, autophagy has been implicated in a variety of human diseases, including cancer, neurodegeneration, and pathogen infection (Shintani and Klionsky, 2004a
). The initial identification of >20 autophagy-related (ATG
) genes in the budding yeast Saccharomyces cerevisiae
has highlighted this single-cell organism as a perfect model to study the molecular mechanism of autophagy, although orthologues of some yeast ATG
genes have been found in higher eukaryotes.
In yeast, autophagy can be induced under starvation conditions to reuse nutrients for essential cellular activities and proper cellular remodeling; this starvation-induced bulk autophagy is considered nonspecific. Studies in yeast have also revealed that S. cerevisiae
has selective autophagic pathways that target specific cargos (Scott et al., 1996
; Hutchins et al., 1999
). These pathways mechanistically and genetically resemble bulk autophagy. One such route is the cytoplasm to vacuole targeting (Cvt) pathway (Nair and Klionsky, 2005
). In this pathway, two vacuolar hydrolases, the precursor form of aminopeptidase I (Ape1 [prApe1]) and α-mannosidase, are transported to the vacuole in a double-membrane vesicle called a Cvt vesicle, with the former subsequently being processed into mature Ape1. Compared with starvation-induced autophagy, the Cvt pathway occurs constitutively in growing conditions. Although some Atg proteins appear to be pathway specific, most are involved in both the specific and nonspecific pathways. However, it is not fully understood how these proteins coordinate and function at the molecular level in either bulk or selective autophagy.
Most yeast Atg components localize at a perivacuolar punctate structure called the preautophagosomal structure (PAS) or phagophore assembly site, which is proposed to be the site of autophagosome and Cvt vesicle formation (Kim et al., 2002
; Noda et al., 2002
). In most endomembrane trafficking systems, such as the early secretory pathway, vesicles form by budding from the surface of a preexisting organelle. However, in autophagy-related processes, the double-membrane sequestering vesicles appear to form de novo; that is, they expand by membrane addition during the formation process rather than being generated from a single piece of contiguous membrane (Reggiori and Klionsky, 2005
). One of the major current challenges is to unveil where the membrane materials for autophagosomes or Cvt vesicles come from and how the lipids are transported to the assembly site. Among all Atg proteins, Atg9 is the best candidate that can help us understand this pivotal issue. Atg9 is the only characterized integral membrane protein required for both autophagosome and Cvt vesicle formation (Noda et al., 2000
). However, this protein is absent from the completed vesicles, suggesting that it is retrieved before the vesicle sealing/completion step. Atg9 localizes to multiple punctate sites, with one of them corresponding to the PAS and others to mitochondria in addition to unidentified structures (Reggiori et al., 2005b
). Recent studies reveal that Atg9 cycles between mitochondria and the PAS vesicle assembly site (Reggiori et al., 2004
). These characteristics make Atg9 a potential membrane carrier for vesicle formation.
We decided to investigate the molecular regulatory mechanisms underlying Atg9 cycling and, in particular, what factors regulate the anterograde transport of Atg9 to the site of vesicle formation. In this study, we discovered that a peripheral membrane protein, Atg11 (Kim et al., 2001
), is an interaction partner of Atg9. The interaction requires the second coiled-coil (CC) domain of Atg11 and the Atg9 N-terminal cytosolic domain. A missense mutation (H192L) in the Atg9 N-terminal domain that disrupts its interaction with Atg11 results in the impaired cycling of Atg9 and a defect in selective autophagy. In addition, we found that in actin mutant cells, Atg11 colocalized with Atg9 and was retained on mitochondria, indicating that Atg11 is not able to direct Atg9 to the PAS in the absence of an intact cytoskeletal network. These data support a model in which a pool of Atg11 links Atg9 to the PAS along the actin cable under vegetative growth conditions.