Vph1-GFP is an unsuitable tool to assess the involvement of ESCRT in microautophagy
To assess microautophagic flux/activity using a processing assay with a GFP-tagged vacuolar membrane protein, its proper localization in vacuolar membranes is prerequisite. First, we assessed whether Vph1 is correctly localized in cells defective in each ESCRT complex, ESCRT-0 to -III, both in normal (nutrient-rich and TORC1 active) conditions and during microautophagy induction after TORC1 inactivation. We found that ESCRT-lacking mutant cells, with mutations in Vps27 (ESCRT-0), Vps28 (ESCRT-I), Vps36 (ESCRT-II) or Vps24 (ESCRT-III), showed massive accumulation of Vph1-GFP in the perivacuolar class E compartment in normal (nutrient-rich and TORC1 active) conditions (Fig. 1, control) as described previously [5, 13-16]. This clearly confirmed that Vph1 is delivered to the vacuolar membrane via the VPS pathway. The aberrant accumulation of Vph1-GFP still remained during microautophagy induction after rapamycin treatment (Fig. 1, +Rap). Namely, Vph1-GFP did not properly reach the vacuolar surface in ESCRT-deficient cells regardless of TORC1 activity. This demonstrated that the Vph1-GFP processing assay is not suitable to evaluate whether ESCRT is directly implicated in microautophagic processes, although Oku et al. proposed it based on results obtained using this assay .
GFP-Pho8 is a suitable marker to evaluate the involvement of ESCRT in microautophagy
Next, we similarly assessed GFP-Pho8. In contrast to most vacuolar proteins trafficked via the VPS pathway, a small number of proteins including the vacuolar membrane alkaline phosphatase (ALP) Pho8 are transported directly from the Golgi to the vacuolar surface, even in class E mutants, via the adaptor protein-3 (AP-3) pathway (or the ALP pathway) [14, 17-19]. We also observed that GFP-Pho8 was appropriately located on vacuolar membranes in ESCRT mutants in normal conditions (Fig. 2, control). Furthermore, its vacuolar localization was not lost during microautophagy induction after cells were treated with rapamycin (Fig. 2, +Rap). Thus, GFP-Pho8 is properly localized on the vacuolar membrane in ESCRT mutants regardless of TORC1 activity. We concluded that GFP-Pho8 is a suitable marker to address the involvement of ESCRT in microautophagy
ESCRT is required for microautophagy after TORC1 inactivation
In a parallel study, we recently reported that free GFP generation from GFP-Pho8 after treatment with rapamycin was massively compromised in cells defective in ESCRT, vps27∆, vps28∆, vps36∆, and vps24∆ cells . Given the proper localization of GFP-Pho8 on vacuolar membranes in these ESCRT mutants, these observations indicated that ESCRT is required for microautophagy itself. To further confirm this we subjected cells to nitrogen starvation, which is a natural condition where TORC1 is inactivated. GFP-Pho8 still showed proper distribution on the vacuolar surface after nitrogen starvation even in ESCRT mutants (Fig. 3a). Autophagic degradation of GFP-Pho8 after nitrogen starvation was mildly reduced in these ESCRT mutants (Fig. 3b) (see “Discussion”). These findings confirmed the idea that ESCRT is indeed required for proper microautophagic induction after TORC1 inactivation.
Search for other appropriate markers of microautophagy
Thus, proteins destined for vacuolar membranes via the AP-3 pathway (the ALP pathway) have potential as appropriate markers to investigate ESCRT-mediated microautophagy. We assessed this using the following vacuolar membrane proteins targeted via the AP-3 pathway: Nyv1 (vacuolar v-SNARE), Yck3 (casein kinase) and Sna4 (protein of unknown function) [20-22]. We found that Nyv1-GFP was not clearly distributed on vacuolar membranes even in wild-type cells (Additional file 1: Fig. 1a). By contrast, clear localization of Yck3-GFP on vacuolar membranes was found in wild-type cells in the absence or presence of rapamycin (Additional file 1: Figs. S1a, b). However, unlike Vph1-GFP and GFP-Pho8, Yck3-GFP did not generate free GFP after rapamycin treatment, although Yck3-GFP was almost lost (Additional file 1: Fig. S1c). This suggested that Yck3 is degraded outside vacuoles (see “Discussion”). Thus, Nyv1-GFP and Yck3-GFP were not suitable for the assessment microautophagic flux. Finally, we found that Sna4-GFP was located on vacuolar membranes in wild-type cells in the absence and presence of rapamycin and that similar vacuolar localization was maintained in ESCRT mutants, although Sna4-GFP signals on the vacuolar membranes were largely lost after rapamycin treatment (Fig. 4, Additional file 1: Fig. S2). Sna4-GFP produced free GFP in wild-type cells after rapamycin treatment (Fig. 5, see also Additional file 1: Fig. S3), which was repressed in vps27∆ cells, similar to GFP-Pho8 . In addition, free GFP generation from Sna4-GFP after rapamycin treatment was also reduced in vps28∆, vps36∆, and vps24∆ cells, although uncharacterized protein bands accumulated additionally in wild-type and the mutant cells. These findings confirmed the hypothesis that ESCRT is required for proper microautophagy induction.