Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole
- Nagoya University Graduate School of Medicine, Japan
Figures
Figure 1 with 3 supplements
Lipophagy in stationary phase occurs through the expansion of raft-like vacuolar membrane domains.
(A) Freeze-fracture EM of the vacuole. The vacuolar membrane in stationary phase yeast shows geometrical patterns in both the protoplasmic face (P face; cytoplasmic leaflet) and the exoplasmic face …
Figure 1—figure supplement 1
The procedure for quick-freezing and freeze-fracture EM.
(A) Quick-freezing and freeze-fracture replica-labeling EM. (a) Quick freezing of live cells without chemical fixation; (b) preparation of freeze-fracture replicas – physical fixation of a …
Figure 1—figure supplement 2
Freeze-fracture/etching EM of an entire yeast cell in stationary phase.
The two rectangular areas are magnified in Figure 1C. Bar, 1 μm.
Figure 1—figure supplement 3
Additional examples of raft-like domain bulging in the vacuole.
Adjacent raft-like domains show variable degrees of bulging. Bars: 0.2 μm.
Figure 2 with 1 supplement
Microautophagic vesicles show the same PtdIns(3)P asymmetry as the vacuolar membrane.
(A, B) Freeze-fracture replica labeling of PtdIns(3)P in stationary phase vacuoles. Colloidal gold particles indicate PtdIns(3)P labeled by recombinant GST-p40phox PX domain. Bars: 0.2 µm. See also F…
Figure 2—figure supplement 1
PtdIns(3)P in a stationary phase vacuole.
(A) Freeze-fracture replica labeling EM of PtdIns(3)P in a stationary phase vacuole. The labeling in the vacuolar membrane was dense in the cytoplasmic leaflet, whereas it was scarce in the …
Figure 3 with 3 supplements
Sterol transport by NPC proteins is essential for stationary phase lipophagy.
(A) Vacuolar domains were classified in three categories: no domain, domains without inward curvature (type I), and domains with inward curvature (type II). Bars: 0.2 µm. In comparison to WT, the …
Figure 3—figure supplement 1
Filipin staining.
(A) Wild-type yeast in log phase was labeled only in the cell surface, but in stationary phase, it showed intense labeling in the vacuolar membrane as well as in the cell surface (arrows), giving …
Figure 3—figure supplement 2
The number and sizes of LDs in stationary phase.
The number and sizes of LDs were measured by fluorescence microscopy after BODIPY493/503 staining and freeze-fracture EM, respectively. Neither of these measurements differed drastically between WT …
Figure 3—figure supplement 3
Microautophagic vesicles in the lumen of a stationary phase vacuole.
(A) Microautophagic vesicles were identified by intense PtdIns(3)P labeling (more than 100 gold labels/μm2) in the cytoplasmic leaflet (colored in red). These were significantly smaller in ncr1Δ and …
Figure 4 with 6 supplements
Trafficking defect of NPC proteins impairs stationary phase lipophagy.
(A) Ncr1p–GFP and Npc2p–GFP in stationary phase were targeted to the vacuole in WT, but showed punctate distribution in a majority of Atg-deficient cells. Bar: 5 µm. The quantification data show the …
Figure 4—figure supplement 1
Time course of Ncr1p–GFP distributional change in WT and mutants.
Ncr1p–GFP in WT distributed to the vacuolar membrane in log phase (day 0) through post-diauxic phase (day 1 and day 2) and stationary phase (day 3). In atg7Δ, Ncr1p–GFP was in the vacuolar membrane …
Figure 4—figure supplement 2
Distribution of Vph1p–mRFP.
Vph1p–mRFP in stationary phase showed similar distribution in the vacuole in WT and atg18Δ. Bar: 5 μm.
Figure 4—figure supplement 3
Filipin staining of atg7Δ in stationary phase.
Filipin staining was performed by the method described for Figure 3E, and the proportion of cells showing the double-ring filipin-staining pattern was measured. The ratio was significantly lower in a…
Figure 4—figure supplement 4
Microautophagic vesicles in atg18Δ with or without expressing Atg18(FTTG)−2xFYVE in stationary phase.
(A) The microautophagic vesicles (colored in red) defined by the PtdIns(3)P labeling in the cytoplasmic leaflet appeared to be of similar sizes in atg18Δ with or without expressing …
Figure 4—figure supplement 5
Freeze-fracture replica EM of fab1Δ in stationary phase.
The vacuolar lumen was vacant. Bar: 0.5 μm.
Figure 4—figure supplement 6
Distribution of Ncr1p–GFP and Npc2p–GFP in stationary phase.
The proportion of cells showing vacuolar distribution of Ncr1p–GFP and Npc2p–GFP was significantly smaller in vps4Δ and nem1Δ than in WT. Mean ± SD of one representative experiment (n > 150 for each …
Figure 5 with 3 supplements
Microautophagy in acute nitrogen starvation also occurs at NPC-dependent raft-like domains.
(A) The IMP-deficient raft-like domains in the vacuolar membrane of WT after nitrogen starvation for 5 hr. These domains were observed both in the cytoplasmic leaflet (P face) and the …
Figure 5—figure supplement 1
Distribution of Vph1p–mRFP in the vacuolar membrane after nitrogen starvation.
Vph1p-mRFP was labeled only in the IMP-rich area (arrows) and not in the IMP-deficient domain. The result verified that the IMP-deficient domain induced by acute nitrogen starvation has raft-like …
Figure 5—figure supplement 2
Close association of the raft-like domain and organelles.
(A) Freeze-fracture EM. LDs and the nucleus were associated with the IMP-deficient domain of the vacuole in nitrogen starvation. LDs were often enclosed within the vacuolar membrane invagination. …
Figure 5—figure supplement 3
Effects of NPC deficiency in nitrogen starvation.
(A) The number and sizes of LDs. Quantification after nitrogen starvation for 5 hr was done by the method described for Figure 3—figure supplement 2. Neither number nor sizes of LDs were drastically …
Figure 6 with 2 supplements
The MVB pathway supplies sterol for induction of microautophagy.
(A) The vacuolar content of ncr1△npc2△ after nitrogen starvation for 5 hr. ILVs, seen as small rugged structures (arrows), and microautophagic vesicles labeled for PtdIns(3)P in the cytoplasmic …
Figure 6—figure supplement 1
Analysis of MVB and vps4Δ.
(A) Freeze-fracture EM of MVB in ypt7Δ. ILVs in the lumen are observed as rugged structures (arrows). Bar: 0.2 μm. (B) Filipin staining in vps4Δ after nitrogen starvation. Vph1p–GFP (green) was …
Figure 6—figure supplement 2
are1Δare2Δ in stationary phase.
(A) Vacuolar domain formation was observed in are1Δare2Δ, although its frequency was lower than that in WT. See Figure 3A for comparison. Mean ± SD of three independent experiments (>50 vacuoles …
Figure 7 with 1 supplement
Involvement of Atg proteins and actin filaments in microautophagy during acute nitrogen starvation.
(A) Raft-like domain formation after nitrogen starvation was significantly less frequent in atg7△ and atg8△ than in WT (see Figure 5B for WT). (B) Degradation of Erg6p–GFP and Nvj1p–GFP in nitrogen …
Figure 7—figure supplement 1
Distribution of Ncr1p–GFP and Npc2p–GFP after nitrogen starvation.
In both WT and atg7Δ, Ncr1p–GFP and Npc2p–GFP were observed in the membrane and in the lumen of the vacuole, respectively. Bar: 5 μm.
Videos
Video 1
Tilted images of a freeze-fracture/etching replica of stationary phase yeast.
An LD revealing the non-etchable content is adhered to the IMP-deficient domain of the vacuolar membrane.
Tables
Table 1
Yeast strains used in this study.
| Name | Genotype | Reference/origin |
|---|---|---|
| SEY6210 | MATα leu2-3,112 ura3-52 his3-∆200 trp1-∆901 lys2-801 suc2-∆9 | Robinson et al. (1988) |
| KVY4 | SEY6210; ypt7Δ::LEU2 | Kihara et al. (2001) |
| KVY5 | SEY6210; atg8Δ::HIS3 | Kirisako et al. (1999) |
| KVY135 | SEY6210; atg7Δ::HIS3 | Obara et al. (2008) |
| TKY1001 | SEY6210; atg18Δ::KanMX | Cheng et al. (2014) |
| YT350 | SEY6210; pep4Δ::hphNT1, prb1Δ::natNT2 | This study |
| YT956 | SEY6210; fab1Δ::cgTRP1 | This study |
| YT1167 | SEY6210; NVJ1–yeGFP::klTRP1 | This study |
| YT1242 | SEY6210; npc2Δ::cgHIS3 | This study |
| YT1276 | SEY6210; ncr1Δ::cgTRP1, npc2Δ::cgHIS3 | This study |
| YT1278 | SEY6210; ncr1Δ::cgTRP1, pho8Δ60::natNT2 | This study |
| YT1287 | SEY6210; VPH1–mRFP::natNT2, ncr1Δ::cgTRP1, npc2Δ::cgHIS3 | This study |
| YT1288 | SEY6210; ncr1Δ::natNT2 | This study |
| YT1296 | SEY6210; VPH1–yeGFP::hphNT1 | This study |
| YT1325 | SEY6210; NCR1–yeGFP::klTRP1 | This study |
| YT1330 | SEY6210; npc2Δ::cgHIS3, pho8Δ60::natNT2 | This study |
| YT1331 | SEY6210; ncr1Δ::cgTRP1, npc2Δ::cgHIS3, pho8Δ60::natNT2 | This study |
| YT1349 | SEY6210; npc2(P143S)::cgTRP1 | This study |
| YT1371 | SEY6210; NPC2–yeGFP::cgTRP1 | This study |
| YT1373 | SEY6210; fab1Δ::cgTRP1, NPC2–yeGFP::hphNT1 | This study |
| YT1385 | SEY6210; atg18Δ::KanMX, NCR1–yeGFP::klTRP1 | This study |
| YT1386 | SEY6210; atg18Δ::KanMX, NPC2–yeGFP::klTRP1 | This study |
| YT1388 | SEY6210; fab1Δ::cgTRP1, NCR1–yeGFP::hphNT1 | This study |
| YT1467 | SEY6210; VPH1–mRFP::natNT2 | This study |
| YT1474 | SEY6210; nem1Δ::cgHIS3, NCR1–yeGFP::klTRP1 | This study |
| YT1476 | SEY6210; nem1Δ::cgHIS3, NPC2–yeGFP::klTRP1 | This study |
| YT1479 | SEY6210; VPH1–yeGFP::hphNT1, ncr1Δ::TRP1, npc2Δ::HIS3 | This study |
| YT1481 | SEY6210; vps4Δ::cgHIS3, NCR1–yeGFP::klTRP1 | This study |
| YT1482 | SEY6210; vps4Δ::cgHIS3, NPC2–yeGFP::klTRP1 | This study |
| YT1503 | SEY6210; atg18Δ::KanMX, ATG18p-atg18(FTTG)−3HA-2FYVE, NCR1–yeGFP::klTRP1 | This study |
| YT1509 | SEY6210; ncr1Δ::cgTRP1, npc2Δ::cgHIS3, ERG6–yeGFP::klTRP1 | This study |
| YT1517 | SEY6210; atg18Δ::KanMX, ATG18p-atg18(FTTG)−3HA-2FYVE, NPC2–yeGFP::klTRP1 | This study |
| YT1523 | SEY6210; ncr1Δ::cgTRP1, npc2Δ::cgHIS3,NVJ1–yeGFP::hphNT1 | This study |
| YT1536 | SEY6210; ncr1Δ::cgTRP1, npc2Δ::cgHIS3, Om45–yeGFP::hphNT1 | This study |
| YT1542 | SEY6210; ERG6–yeGFP::natNT2 | This study |
| YT1549 | SEY6210; vps4Δ::natNT2, NVJ1–yeGFP::hphNT1 | This study |
| YT1552 | SEY6210; atg7Δ::HIS3, NCR1–yeGFP::klTRP1 | This study |
| YT1553 | SEY6210; atg3Δ::KanMX, NCR1–yeGFP::klTRP1 | This study |
| YT1554 | SEY6210; atg5Δ::LEU2, NCR1–yeGFP::klTRP1 | This study |
| YT1556 | SEY6210; atg2Δ::LEU2, NCR1–yeGFP::klTRP1 | This study |
| YT1557 | SEY6210; atg1Δ::KanMX, NCR1–yeGFP::klTRP1 | This study |
| YT1558 | SEY6210; atg7Δ::HIS3, NPC2–yeGFP::klTRP1 | This study |
| YT1559 | SEY6210; atg3Δ::KanMX, NPC2–yeGFP::klTRP1 | This study |
| YT1560 | SEY6210; atg5Δ::LEU2, NPC2–yeGFP::klTRP1 | This study |
| YT1561 | SEY6210; atg8Δ::HIS3, NPC2–yeGFP::klTRP1 | This study |
| YT1562 | SEY6210; atg2Δ::LEU2, NPC2–yeGFP::klTRP1 | This study |
| YT1569 | SEY6210; atg1Δ::KanMX, NPC2–yeGFP::klTRP1 | This study |
| YT1579 | SEY6210; atg8Δ::HIS3, NCR1–yeGFP::klTRP1 | This study |
| YT1583 | SEY6210; vps4Δ::cgHIS3, ERG6–yeGFP::klTRP1 | This study |
| YT1584 | SEY6210; atg8Δ::HIS3, NVJ1–yeGFP::klTRP1 | This study |
| YT1586 | SEY6210; atg7Δ::HIS3, ERG6–yeGFP::klTRP1 | This study |
| YT1591 | SEY6210; atg14Δ::LEU2, ERG6–yeGFP::natNT2 | This study |
| YT1592 | SEY6210; atg14Δ::LEU2, NVJ1–yeGFP::natNT2 | This study |
| YT1597 | SEY6210; atg8Δ::HIS3, ERG6–yeGFP::klTRP1 | This study |
| YT1598 | SEY6210; atg7Δ::HIS3, NVJ1–yeGFP::klTRP1 | This study |
| YT1726 | SEY6210; pep4Δ::hphNT1, prb1Δ::natNT2, atg7Δ::HIS3 | This study |
