Evidence for ESCRT- and clathrin-dependent microautophagy - PubMed (original) (raw)

Evidence for ESCRT- and clathrin-dependent microautophagy

Masahide Oku et al. J Cell Biol. 2017.

Abstract

Microautophagy refers to a mode of autophagy in which the lysosomal or vacuolar membrane invaginates and directly engulfs target components. The molecular machinery of membrane dynamics driving microautophagy is still elusive. Using immunochemical monitoring of yeast vacuolar transmembrane proteins, Vph1 and Pho8, fused to fluorescent proteins, we obtained evidence showing an induction of microautophagy after a diauxic shift in the yeast Saccharomyces cerevisiae Components of the endosomal sorting complex required for transport machinery were found to be required for this process, and the gateway protein of the machinery, Vps27, was observed to change its localization onto the vacuolar membrane after a diauxic shift. We revealed the functional importance of Vps27's interaction with clathrin in this microautophagy that also contributed to uptake of lipid droplets into the vacuole. This study sheds light on the molecular mechanism of microautophagy, which does not require the core Atg proteins.

© 2017 Oku et al.

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Figures

Figure 1.

Figure 1.

Vacuolar dynamics after diauxic shift. (A) Immunoblot analyses of Vph1-EGFP and yEGFP-Pho8 expressed in either the WT or _pep4_Δ strain. Cells were taken from cultures in YEPD medium at 28°C for the indicated time period and subjected to immunoblot analysis with an anti-GFP antibody. FL represents a band position for the full-length form of the fusion proteins, and CL indicates that for the c

l

eaved form containing EGFP or yEGFP moiety. The immunoblot data gained with anti–β-actin antibody are shown as loading controls. (B) Time course of glucose consumption (open circle) and growth (closed circle) of the WT strain in YEPD medium. The mean values from three independent cultures are plotted, and error bars indicate standard deviation. (C) A representative EM image of a WT strain cultured in YEPD medium at 28°C for 16 h. The vacuole invaginations are highlighted with black arrows. V, vacuole. Bar, 0.5 µm.

Figure 2.

Figure 2.

Immunoblot analyses detecting cleavage of Vph1-EGFP expressed in mutant strains defective in Atg pathways. The denoted ATG gene–deleted mutant strains (A) or other gene-deletion mutants (B) were cultured at 28°C for 24 h in YEPD medium and subjected to immunoblot analysis as shown in Fig. 1 A.

Figure 3.

Figure 3.

Functional analysis of domain-mutated Vps27 derivatives. (A) Schematic drawing of Vps27 intramolecular domains. The binding partner of each domain is also shown. CB, clathrin-binding motif; PTAPL, PTAP-like amino-acid sequence. (B) Immunoblot analysis of Vph1-EGFP in vps27 mutants. The data are presented as in Fig. 1 A. In the _VPS27_-deleted (_vps27_Δ) strain, normal (WT) or mutant forms of Vps27 were expressed. The designation of mut indicates introduction of point mutations in the denoted domains. The cleavage rate for each variant [(band intensity of CL)/(band intensities of FL + CL) × 100 (%)] was calculated from three independent experiments (right). The error bars indicate standard deviation. (C) Immunoblot analysis of yEGFP-Pho8 expressed in vps27 mutant strains. The asterisk indicates a nonspecific band. (D) Immunoblot analysis of Vph1-EGFP expressed in chc1 (clathrin heavy chain) mutant or WT strain cultured at 23°C for 24 h (23°C) or cultured at 23°C for 16 h and subsequently cultured at 31°C for 8 h (31°C). The cleavage rates calculated as in B from three independent experiments are also shown. Error bars indicate standard deviation.

Figure 4.

Figure 4.

Localizations of Vps27 variants during different growth phases. _VPS27_-deleted (_vps27_Δ) strains expressing Envy-tagged WT (A), FYVE-domain–mutated Vps27 (B), or clathrin-binding motif–deleted Vps27 (C) were cultured in YEPD medium at 28°C for 8 h (at an early log phase) or 14 h (after diauxic shift). Both cells labeled with FM 4–64 and nonlabeled cells were analyzed by fluorescence microscopy with the same image-acquisition parameters. The merge image consists of green Envy image and red FM 4–64 image. Bars, 2 µm. (D) The numbers of cells exhibiting diffuse, punctate, and/or rim patterns of Envy-Vps27 were counted (n > 50) for each observation, and the percentages of the patterns are represented in the stacked bar chart. Error bars indicate standard deviation.

Figure 5.

Figure 5.

Efficient transport of LDs into the vacuole in cells after the diauxic shift depends on Vps27 harboring the clathrin-binding motif. (A) Immunoblot analysis of Osw5-EGFP in the designated mutant strains cultured in YEPD medium at 28°C for 16 h. The data for anti–actin antibody are also shown as a loading control. CL; cleaved form; FL, full-length form;. The graph indicates the cleavage rates calculated from three independent experiments. The error bars indicate standard deviation, and the double asterisk indicates statistical significance (**, P < 0.01, _t_ test). (B) The numbers of LDs found inside the vacuoles in EM images of the designated strains (_n_ > 50) were compared. Error bars indicate the standard error. The asterisk indicates the statistical significance of the comparison (*, P < 0.05, t test).

Figure 6.

Figure 6.

Schematic model of microautophagy proposed in this study.

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