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. 2007 Jan;18(1):106-18.
doi: 10.1091/mbc.e06-06-0554. Epub 2006 Nov 1.

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers

Taras Y Nazarko  1 , Andriy S Polupanov, Ravi R Manjithaya, Suresh Subramani, Andriy A Sibirny
Affiliations

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers

Taras Y Nazarko et al. Mol Biol Cell. 2007 Jan.

Abstract

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repeat protein is not essential for macropexophagy of methanol-, oleate-, or amine-induced peroxisomes, which makes PpVac8 the first known protein required for the micropexophagy, but not for the macropexophagy, machinery. The uniqueness of Atg26 and Vac8 functions under different pexophagy conditions demonstrates that not only pexophagy inducers, such as glucose or ethanol, but also the inducers of peroxisomes, such as methanol, oleate, or primary amines, determine the requirements for subsequent pexophagy in yeast.

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Figures

Figure 1.
Figure 1.
The Y. lipolytica ATG26 gene is not required for selective degradation of oleate- and amine-induced peroxisomes. Y. lipolytica WT (H222), atg26 (N155), and trs85-2 (Ain19) cells were induced in (A) oleate/ammonia or (B) (−C)/methylamine medium and transferred to glucose/(−N) medium to induce pexophagy. At the indicated time points, the culture samples were collected and processed for immunoblotting with antibodies against peroxisomal THI and mitochondrial Hsp60 proteins as described in Materials and Methods.
Figure 2.
Figure 2.
Oleate- and amine-induced peroxisomes in Y. lipolytica are delivered to the vacuoles by macropexophagy. (A) Y. lipolytica WT (STN001) cells were induced in oleate/ammonia medium and transferred to glucose/(−N) medium with FM 4-64. (B) Y. lipolytica WT (STN002) cells were induced in (−C)/methylamine medium with FM 4-64 and transferred to glucose/(−N) medium. At the indicated time points, the progress of pexophagy was determined by fluorescence microscopy as described in Materials and Methods. Peroxisomes were labeled with Aox3-EYFP and vacuolar membranes with FM 4-64. Bar, 5 μm.
Figure 3.
Figure 3.
Macropexophagy of oleate- and amine-induced peroxisomes in Y. lipolytica does not depend on Atg26. (A) Y. lipolytica WT (STN001), atg26 (STN003), and trs85-1 (STN012) cells were induced in oleate/ammonia medium and transferred to glucose/(−N) medium. (B) Y. lipolytica WT (STN001), atg26 (STN003), and trs85-1 (STN011) cells were induced in (−C)/methylamine medium and transferred to glucose/(−N) medium. After 9 h, the progress of pexophagy was determined by fluorescence microscopy. Peroxisomes were labeled with Aox3-EYFP. Bar, 5 μm.
Figure 4.
Figure 4.
The P. pastoris ATG26 gene is not essential for selective degradation of oleate- and amine-induced peroxisomes, but it increases its efficiency. P. pastoris WT (GS200), Δatg26 (Pdg3D), and pep4 prb1 (SMD1163) cells were induced in oleate/ammonia (A), methanol/ammonia (B), (−C)/methylamine (C), or methanol/(−N) (D) medium and transferred to glucose/(−N) medium to induce pexophagy. At the indicated time points, the culture samples were collected and processed for immunoblotting with antibodies against peroxisomal THI or AOX.
Figure 5.
Figure 5.
Oleate- and amine-induced peroxisomes in P. pastoris are delivered to the vacuoles by macropexophagy. (A) P. pastoris WT (STN018) cells were induced in oleate/ammonia medium with FM 4-64 and transferred to glucose/(−N) medium. (B) P. pastoris WT (STN026) cells were induced in (−C)/methylamine medium with FM 4-64 and transferred to glucose/(−N) medium. The progress of pexophagy was determined by fluorescence microscopy. Peroxisomes were labeled with GFP-SKL and vacuolar membranes with FM 4-64. Bar, 5 μm.
Figure 6.
Figure 6.
Macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris can proceed without the ATG26 gene but at slower rates. (A) P. pastoris WT (STN018), Δatg26 (STN020), and atg1-1 (STN022) cells were induced in oleate/ammonia medium and transferred to glucose/(−N) medium for 9 h. (B) P. pastoris WT (STN026), Δatg26 (STN028), and atg1-1 (STN029) cells were induced in (−C)/methylamine medium and transferred to glucose/(−N) medium for 3 h. The progress of pexophagy was determined by fluorescence microscopy. Peroxisomes were labeled with GFP-SKL. Bar, 5 μm.
Figure 7.
Figure 7.
The P. pastoris ATG26 but not the VAC8 gene is essential for macropexophagy of methanol-induced peroxisomes during ethanol adaptation. The P. pastoris WT (GS200), Δatg26 (Pdg3D), Δvac8 (WDY53), and pep4 prb1 (SMD1163) cells were induced in methanol/ammonia (A) or methanol/(−N) (B) medium and transferred to ethanol/(−N) medium to induce pexophagy. At the indicated time points, the culture samples were collected and processed for immunoblotting with antibodies against peroxisomal AOX. (C) The P. pastoris WT (STN026), Δatg26 (STN028), Δvac8 (STN032), and atg1-1 (STN030) cells were induced in methanol/ammonia medium with FM 4-64 and transferred to ethanol/(−N) medium for 3 h. Pexophagy was monitored by fluorescence microscopy. Peroxisomes were labeled with GFP-SKL and vacuolar membranes with FM 4-64. Bar, 5 μm.
Figure 8.
Figure 8.
The P. pastoris VAC8 gene is not essential for degradation of oleate- and amine-induced peroxisomes. P. pastoris WT (GS200), Δvac8 (WDY53), and pep4 prb1 (SMD1163) cells were induced in oleate/ammonia (A) or (−C)/methylamine (B) medium and transferred to glucose/(−N) medium to induce pexophagy. At the indicated time points, the culture samples were collected and processed for immunoblotting with antibodies against peroxisomal THI or AOX. (C) P. pastoris WT (STN018), Δvac8 (STN024), and atg1-1 (STN022) cells were induced in oleate/ammonia medium and transferred to glucose/(−N) medium for 9 h. (D) P. pastoris WT (STN026), Δvac8 (STN031), and atg1-1 (STN029) cells were induced in (−C)/methylamine medium and transferred to glucose/(−N) medium for 3 h. The progress of pexophagy was determined by fluorescence microscopy. Peroxisomes were labeled with GFP-SKL. Bar, 5 μm.
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