Endocytosis in S. cerevisiae was mainly regulated by two Rab modules (Vps21 and Ypt7 modules, respectively, each consists of upstream regulators, Rab, and down stream effectors). In addition, the Ypt7 module functions in the fusion of mature autophagosomes with vacuoles in autophagy. We recently reported that the Vps21 module also plays important roles in autophagy. In the absence of Vps21 module proteins, the autophagosomes accumulated outside the vacuoles to be like a crescent. We further investigated the character of the autophagosomes in Vps21 module mutants. Proteinase protection assay showed that the autophagosomes in the Vps21 (and Ypt7) module mutants were unclosed autophagosomes, different from the closed autophagosomes in the Ypt7 module mutants alone. Atg protein localization experiments further support the character of unclosed autophagosomes in the Vps21 module mutants as most Atg proteins colocalized with autophagosomes in a crescent in them, while most Atg proteins released from the autophagosomes in the Ypt7 module mutants. Based on the interactions between the Vps21 module and the PI3K complexes, and the mislocalized PI3K complex and its product PI3P in the Vps21 module mutants, we proposed that the Vps21 module-dependent localization of the PI3K complex is important for the functions of the Vps21 module proteins in autophagosome closure, in front of the Ypt7 module proteins in autophagosome fusion with the vacuoles.