PpAtg30 tags peroxisomes for turnover by selective autophagy

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.     

Pexophagy: autophagic degradation of peroxisomes

The abundance of peroxisomes within a cell can rapidly decrease by selective autophagic degradation (also designated pexophagy). Studies in yeast species have shown that at least two modes of peroxisome degradation are employed, namely macropexophagy and micropexophagy. During macropexophagy, peroxisomes are individually sequestered by membranes, thus forming a pexophagosome. This structure fuses with the vacuolar membrane, resulting in exposure of the incorporated peroxisome to vacuolar hydrolases. During micropexophagy, a cluster of peroxisomes is enclosed by vacuolar membrane protrusions and/or segmented vacuoles as well as a newly formed membrane structure, the micropexophagy-specific membrane apparatus (MIPA), which mediates the enclosement of the vacuolar membrane. Subsequently, the engulfed peroxisome cluster is degraded. This review discusses the current state of knowledge of pexophagy with emphasis on studies on methylotrophic yeast species.     

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Role of Vac8 in Cellular Degradation Pathways in Pichia pastoris

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy and mitophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity and GFP-labeled mitochondria in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like “segmented” extensions that almost completely surrounded the adjacent peroxisomes. PpVac8-GFP was found at the vacuolar membrane and concentrated at the base of the sequestering membranes that extend from the vacuole to engulf the peroxisomes. The localization of PpVac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8, resulted in an impaired vacuolar association and decreased degradation of alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a nonfunctional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.     

Roles of Pichia pastoris Uvrag in vacuolar protein sorting and the phosphatidylinositol 3-kinase complex in phagophore elongation in autophagy pathways

Although it has been established that Atg6/Beclin 1, the phosphatidylinositol 3-kinase (PI3K) Vps34, and associated proteins have direct or indirect roles in autophagic pathways in both mammals and yeasts, the elucidation of these roles and the proteins required for them is ongoing. The involvement of the Beclin 1-binding protein, UVRAG, has been a particular source of disagreement. We found that PpAtg6 is required for all autophagic pathways that have been identified in the yeast Pichia pastoris, as well as for the carboxypeptidase Y (PpCPY) vacuolar protein sorting pathway. We localized PpAtg6 to the phagophore assembly site (PAS) and observed its continued presence at that site as the isolation membrane grew from it and matured into a pexophagosome. PpUvrag, however, was required for proper PpCPY sorting, but not for any autophagic pathway. Rather, the defects in all autophagic pathways observed when PpUvrag was overexpressed support its presence in a complex that competes with the PI3K complex required for autophagy.     

Role of Vac8 in Formation of the Vacuolar Sequestering Membrane during Micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.     

Functions of PI4P and sterol glucoside are necessary for the synthesis of a nascent membrane structure during pexophagy

We recently showed that, in the yeast Pichia pastoris, an ergosterol glucoside synthesizing enzyme, Atg26, is recruited to the precursor of the pexophagic structure, micropexophagic membrane apparatus (MIPA), under the regulation of phosphatidylinositol 4'-monophosphate (PI4P)-signaling during pexophagy. Atg26 was found to harbor a novel PI4P-binding motif, the GRAM domain. Both lipids, PI4P and sterol glucoside, synthesized by PpPik1 and PpAtg26, respectively, were necessary for pexophagy, in the step where the MIPA was formed. In this addendum, we review these findings, and speculate on the mechanistic and physiological implications of the functions of these lipids during the autophagic process.     

PI4P-signaling pathway for the synthesis of a nascent membrane structure in selective autophagy

Phosphoinositides regulate a wide range of cellular activities, including membrane trafficking and biogenesis, via interaction with various effector proteins that contain phosphoinositide binding motifs. We show that in the yeast Pichia pastoris, phosphatidylinositol 4'-monophosphate (PI4P) initiates de novo membrane synthesis that is required for peroxisome degradation by selective autophagy and that this PI4P signaling is modulated by an ergosterol-converting PpAtg26 (autophagy-related) protein harboring a novel PI4P binding GRAM (glucosyltransferase, Rab-like GTPase activators, and myotubularins) domain. A phosphatidylinositol-4-OH kinase, PpPik1, is the primary source of PI4P. PI4P concentrated in a protein-lipid nucleation complex recruits PpAtg26 through an interaction with the GRAM domain. Sterol conversion by PpAtg26 at the nucleation complex is necessary for elongation and maturation of the membrane structure. This study reveals the role of the PI4P-signaling pathway in selective autophagy, a process comprising multistep molecular events that lead to the de novo membrane formation.     

Role of Vac8 in formation of the vacuolar sequestering membrane during micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.     

Molecular mechanism and physiological role of pexophagy

Pexophagy is a selective autophagy process wherein damaged and/or superfluous peroxisomes undergo vacuolar degradation. In methylotropic yeasts, where pexophagy has been studied most extensively, this process occurs by either micro- or macropexophagy: processes analogous to micro- and macroautophagy. Recent studies have identified specific factors and illustrated mechanisms involved in pexophagy. Although mechanistically pexophagy relies heavily on the core autophagic machinery, the latest findings about the role of auxiliary pexophagy factors have highlighted specialized membrane structures required for micropexophagy, and shown how cargo selectivity is achieved and how cargo size dictates the requirement for these factors during pexophagy. These insights and additional observations in the literature provide a framework for an understanding of the physiological role(s) of pexophagy.     

Peroxisomicine A1, a potential antineoplastic agent,causes micropexophagy in addition to macropexophagy

Peroxisomicine A1 (PA1) is a potential antineoplastic agent with high and selective toxicity toward peroxisomes of tumor cells. Pexophagy is a selective autophagy process that degrades damaged peroxisomes; this process has been studied mainly in methylotrophic yeasts. There are two main modes of pexophagy in yeast: macropexophagy and micropexophagy. Previous studies showed that peroxisomes damaged by a prolonged exposition to PA1 are eliminated by macropexophagy. In this work, Candida boidinii was grown in methanol‐containing media, and PA1 was added to the cultures at 2 µg/mL after they reached the mid‐exponential growth phase. Samples were taken at 5, 10, 15, 20, and 25 min after the addition of PA1 and processed for ultrastructural analysis. Typical morphological characteristics of micropexophagy were observed: the direct engulfment of peroxisomes by the vacuolar membrane and the presence of the micropexophagic membrane apparatus (MIPA), which mediates the fusion between the opposing tips of the vacuole to complete sequestration of peroxisomes from the cytosol. In conclusion, here we report that, in addition to macropexophagy, peroxisomes damaged by PA1 can be eliminated by micropexophagy. This information is useful to deepen the knowledge of the mechanism of action of PA1 and of that of pexophagy per se.     

Atg18 lifts up from and lands on the vacuolar membrane mediated by phosphorylation of its propellers

Pichia pastoris Atg18 (PpAtg18), a member of the PROPPIN family of proteins, is localized not only to the PAS (pre-autophagosomal structure or phagophore assembly site) during autophagy but also to the vacuolar membrane during vacuolar fission. Recently we reported that the localization of Atg18 was determined by its phosphorylation level. We identified two phosphorylated regions within the β-propeller structures of PpAtg18, whose modification affects its affinity toward phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂]. The findings indicated that phosphoregulaton of Atg18 mediates the signal from various environmental stimuli and regulates its intracellular localization for vacuolar fission and autophagy.     

Functions of PI4P and Sterol Glucoside Necessary for the Synthesis of a Nascent Membrane Structure During Pexophagy

We recently showed that, in the yeast Pichia pastoris, an ergosterol glucoside synthesizing enzyme Atg26 is recruited to the precursor of the pexophagic structure, micropexophagic membrane apparatus (MIPA), under the regulation of phosphatidylinositol 4'-monophosphate (PI4P)-signaling during pexophagy. Atg26 was found to harbor a novel PI4P-binding motif, GRAM domain. Both lipids, PI4P and sterol glucoside, synthesized by PpPik1 and PpAtg26, respectively, were necessary for pexophagy, in the step where the MIPA was formed. In this addendum, we review these findings, and speculate the mechanistic and physiological implications of the functions of these lipids during the autophagic process.

Addendum to:

PI4P-Signaling Pathway for the Synthesis of a Nascent Membrane Structure in Selective Autophagy

S. Yamashita, M. Oku, Y. Wasada, Y. Ano and Y. Sakai

J Cell Biol 2006; 173: 709-17     

Hansenula polymorpha Vam7p is required for macropexophagy

We have analyzed the functions of two vacuolar t-SNAREs, Vam3p and Vam7p, in peroxisome degradation in the methylotrophic yeast Hansenula polymorpha. A Hp-vam7 mutant was strongly affected in peroxisome degradation by selective macropexophagy as well as non-selective microautophagy. Deletion of Hp-Vam3p function had only a minor effect on peroxisome degradation processes. Both proteins were located at the vacuolar membrane, with Hp-Vam7p also having a partially cytosolic location. Previously, in baker's yeast Vam3p and Vam7p have been demonstrated to be components of a t-SNARE complex essential for vacuole biogenesis. We speculate that the function of this complex in macropexophagy includes a role in membrane fusion processes between the outer membrane layer of sequestered peroxisomes and the vacuolar membrane. Our data suggest that Hp-Vam3p may be functionally redundant in peroxisome degradation. Remarkably, deletion of Hp-VAM7 also significantly affected peroxisome biogenesis and resulted in organelles with multiple, membrane-enclosed compartments. These morphological defects became first visible in cells that were in the mid-exponential growth phase of cultivation on methanol, and were correlated with accumulation of electron-dense extensions that were connected to mitochondria.     

Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle

Three membrane microdomains can be identified on docked vacuoles: "outside" membrane, not in contact with other vacuoles, "boundary" membrane that contacts adjacent vacuoles, and "vertices," where boundary and outside membrane meet. In living cells and in vitro, vacuole fusion occurs at vertices rather than from a central pore expanding radially. Vertex fusion leaves boundary membrane within the fused organelle and is an unexpected pathway for the formation of intralumenal membranes. Proteins that regulate docking and fusion (Vac8p, the GTPase Ypt7p, its HOPS/Vps-C effector complex, the t-SNARE Vam3p, and protein phosphatase 1) accumulate at these vertices during docking. Their vertex enrichment requires cis-SNARE complex disassembly and is thus part of the normal fusion pathway.     

Fusion Proteins and Select Lipids Cooperate as Membrane Receptors for the Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Vam7p

Vam7p, the vacuolar soluble Q_c-SNARE, is essential for yeast vacuole fusion. The large tethering complex, homotypic fusion and vacuole protein sorting complex (HOPS), and phosphoinositides, which interact with the Vam7p PX domain, have each been proposed to serve as its membrane receptors. Studies with the isolated organelle cannot determine whether these receptor elements suffice and whether ligands or mutations act directly or indirectly on Vam7p binding to the membrane. Using pure components that are active in reconstituted vacuolar fusion, we now find that Vam7p binds to membranes through its combined affinities for several vacuolar membrane constituents: HOPS, phosphatidylinositol 3-phosphate, SNAREs, and acidic phospholipids. Acidic lipids allow low concentrations of Vam7p to suffice for fusion; without acidic lipids, the block to fusion is partially bypassed by high concentrations of Vam7p.     

PpAtg9 trafficking during micropexophagy in Pichia pastoris

PpAtg9 is essential for the selective degradation of peroxisomes (e.g., pexophagy) in Pichia pastoris. This integral membrane protein is synthesized in the endoplasmic reticulum (ER) and transported to a unique peripheral compartment (Atg9-PC). A putative ER exit motif has been identified and when deleted results in the accumulation of PpAtg9 within the ER. Upon the onset of micropexophagy, PpAtg9 transits from the Atg9-PC to perivacuolar structures (PVS) and sequestering membranes (SM) that arise from the vacuole to engulf the peroxisomes. In this article, we will discuss the transport pathways of PpAtg9 and those factors responsible for its trafficking.     

Atg28, a novel coiled-coil protein involved in autophagic degradation of peroxisomes in the methylotrophic yeast Pichia pastoris

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy. We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.     

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

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.