Microautophagy (MI-autophagy) is an umbrella term for intracellular degradative pathways that entail the invagination or protrusion of the limiting membranes of endolysosomal compartments, that is, late endosomes and mammalian lysosomes or yeast and plant vacuoles, followed by pinching-off of the membrane into the lumen of the organelle. During these processes, the material specifically and nonspecifically targeted for degradation is sequestered within the invaginating or protuberating membrane. In contrast to macroautophagy, the molecular mechanisms underlying MI-autophagy are largely unknown due to their diversity and complexity in location, regulation and molecular machinery requirements. Here, we review recent progress in the field of MI-autophagy, describing the molecular basis and functions of the MI-autophagic pathways reported to date in eukaryotic cells, from yeast to mammalian and plant cells.

Nucleus-vacuole junctions (NVJs) in Saccharomyces cerevisiae serve as an inter-organellar hub for multiple cell processes, including lipid transport and biosynthesis, an intra-nuclear quality control mechanism, and piecemeal microautophagy of the nucleus. NVJs are formed by complexes between Vac8 in the vacuole membrane and Nvj1 in the nuclear envelope. Nvj1 also links the inner and outer nuclear membranes across the perinuclear lumen. Orthologs of Nvj1 had previously only been found in yeasts of the order Saccharomycetales, raising the possibility that NVJs are restricted to this clade. Using homology and synteny, we discovered scores of novel Nvj1 orthologs across 200 million years of evolution within the subphylum Saccharomycotina. Not all orthologs mediate NVJ formation when expressed in S. cerevisiae, and some lack a sequence motif necessary for this function. Furthermore, the sequence motif required for binding the oxysterol binding protein Osh1 and an associated novel sequence motif, both found only in the order Saccharomycetales, were independently lost in three different lineages. This collection suggests specific opportunities to explore evolutionary and functional adaptations of Nvj1s in ecologically and physiologically diverse yeasts.

Atg18, Atg21 and Hsv2 are homologous proteins that fulfill macroautophagic/autophagic and non-autophagic functions. We now found that Atg21 interacts with Pep8/Vps26, Vps29 and Vps35, the components of the cargo selective complex of the retromer. We identified Atg21 residues required for retromer binding and focused on two of them. The first, T106, is part of an STS-motif, which also mediates Atg18-binding to the retromer, while in Hsv2 this motif is not conserved. As a second retromer binding residue, we identified D28 of Atg21. Interestingly, the corresponding D45 of Hsv2 also confers retromer binding, but the analogous E34 of Atg18 does not. Together, Atg18 uses binding residue 1, while Atg21 uses 1 and 2 and Hsv2 only 2. During autophagy, Atg21 organizes the Atg8-lipidation machinery by interacting with Atg16 via the bottom side of its β-propeller. Partial overlap between the Atg16 binding residues and the retromer binding residues indicates mutually exclusive interaction. Indeed, lack of Atg16 enhances Atg21 binding to the retromer. The Atg21-retromer shows vacuole fission activity, which requires both retromer binding residues and the membrane-bending activity of its loop 6 C/D. Additionally, overexpression of Atg21 led to mislocalization of the Prc1/carboxypeptidase Y cargo receptor Pep1/Vps10 from the Golgi to Vps17-positive endosomes and to Prc1 secretion. We detected a cross-talk among the different retromer complexes. In the absence of the canonical retromer component Vps5, more Atg21-retromer complexes were formed. Furthermore, the vacuole hyper-fragmentation of vps17Δ cells cooperatively required Atg18 and Atg21. Along this line, we found that Atg21 interacts with Atg18 and Hsv2.Abbreviation: Atg: autophagy related, CSC: cargo specific complex (of the retromer), PAS: phagophore assembly site, Prc1/CPY/carboxypeptidase Y: proteinase C, PROPPIN: beta-propeller that binds phosphoinositides.

Autophagosome formation depends on PtdIns3P, its presence is deciphered by PROPPINs, a family of β-propellers, which in yeast consists of Atg18, Atg21 and Hsv2 and in mammals of WIPI1, WIPI2, WDR45B/WIPI3 and WDR45/WIPI4. While Atg18 is required for scaffolding the Atg2-Atg9 complex, which mediates non-vesicular membrane transport to the phagophore, Atg21 organizes the Atg8 lipidation machinery. Atg18 further acts as part of a retromer complex in vacuole fragmentation. So far, the function of Hsv2 remained elusive. Here we show that Hsv2 is required for autophagy of large cargos such as the fatty acid synthase complex (FAS) and ribosomes. We further found that Hsv2 interacts with the key retromer component Vps35 and mediates vacuole fission cooperatively with Atg18. Interestingly, the residues for interaction of Hsv2 with Atg2 and Vps35 are distinct from those of Atg18. Hsv2 is known to affect the biogenesis of the spore wall, which prompted us to include diploid cells in our analyses. We found that Hsv2 interacts with the SNARE Pep12, and that Pep12 mislocalized to the vacuole in diploid but not haploid hsv2∆ cells. This suggests a role of Hsv2 in protein sorting in diploid cells. The loop 6C/D of PROPPINs partially inserts into membranes causing their bending. We found that the membrane bending activity of Hsv2 is required for vacuole fragmentation and sorting in diploids but not for its autophagic function. Mutations in WDR45/WIPI4, the presumed mammalian homolog of Hsv2 cause the neurodegenerative disease BPAN, our study thus also helps to understand its underlying principles.Abbreviations: CSC: cargo specific complex, FAS: fatty acid synthase, PAS: phagophore assembly site; PROPPIN: beta-propeller that binds phosphoinositides.

Recently, rapid progress in the field of microautophagy (MI-autophagy) revealed the existence of multiple subtypes that differ in both intracellular membrane dynamics and molecular mechanisms. As a result, a single umbrella term "microautophagy" has become too vague, even creating some confusion among researchers both within and outside the field. We herein describe different subtypes of MI-autophagic processes and propose a systematic approach for naming them more accurately.Abbreviation: ATG, autophagy related; e-MI, endosomal microautophagy; ER, endoplasmic reticulum; ESCRT, endosomal sorting complex required for transport; EV, extracellular vesicle; HSPA8/HSC70, heat shock protein family A (Hsp70) member 8; ILVs, intralumenal vesicles; l-MI, lysosomal microautophagy; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MCOLN1, mucolipin TRP cation channel 1; microautophagy, MI-autophagy; MVBs, multivesicular bodies; SQSTM1, sequestosome 1; v-MI, vacuolar microautophagy.

The goals and scope of many prestigious journals today encourage authors to submit articles in areas of great biological importance, and autophagy is often and explicitly mentioned here. This has not always been the case. For a long time, the question of how excess or damaged intracellular material is transported to lysosomes/vacuoles for degradation and reuse only interested a few experts studying this macroautophagic (hereafter autophagic) process. Thirty years ago, the pioneering autophagy expert Yoshinori Ohsumi published the identification of the first yeast autophagy genes in FEBS Letters [[1]] and received the Nobel Prize in Physiology or Medicine 2016 for this groundbreaking discovery. The fact that the discovery of autophagy genes has not only opened the door to the molecular understanding of autophagy [[2]] but has also sparked a strong interest in this ancient cellular mechanism in eukaryotes, can be seen in the astonishing number of studies published on autophagy since 1993. A brief search of the first 30 years of autophagy research from 1950 to 1993 yielded below 500 publications, but of the last 30 years from 1993 to 2023, there are over 80 000. This still growing interest in autophagy coupled with the 30th anniversary of the discovery of the first autophagy mutants in yeast prompted us to create this special issue of FEBS Letters. Here, in the 12 review articles, internationally recognized experts in the field of autophagy research describe important aspects in connection with the pioneering work in the field of autophagy and highlight current findings in relation to the molecular and functional understanding of the autophagy process. The first verifiable report of the use of the term autophagy can be found way back in 1859 [[3]]. However, it was only much later, when Christian de Duve first used the term autophagy in 1963 to describe a lysosomal degradation pathway for cell's own material that the foundation for research into the mechanism of autophagy was laid [[4]]. Back then, the study of autophagy was predominantly based on morphological analysis, for which the use of electron microscopes was of unique importance. Also today, morphological analysis of the various autophagic structures is an essential tool of choice to understand the process more in depth. Recent groundbreaking developments toward super-resolution power of both light and electron microscopy, as well as their combined application, have catapulted research into the formation and maturation of autophagosomes enormously forward [[5]]. In this context, Eskelinen [[6]] here highlights new findings in autophagosome biogenesis through the use of cryo-electron tomography in her expert review. Early on, from the 1960s onwards, biochemical modulators were used to study the process of autophagy. It turned out that the formation of autophagosomes requires the production of phosphatidylinositol-3-phosphate (PI3P), is controlled by metabolic hormones, and that amino acids inhibit autophagy [[7-11]]. On this basis, the intracellular targets were identified, TOR and AMPK, which respond to the large number of different signaling pathways and control the initiation of autophagy [[12]]. These principal signaling pathways are conserved, and the review by Patrice Codogno and colleagues [[13]] provides a historical overview of autophagy control in mammalian systems, and Metur and Klionsky [[14]] address nutrient-controlled signaling pathways in the yeast Saccharomyces cerevisiae. Early pioneering work showed that there are also forms of autophagy in which cytoplasmic material is directly transported into the lysosome of mammalian cells or the yeast vacuole without the cargo being initially sequestered in an autophagosome. Since then, microautophagy has described the lysosomal degradation of small portions of cytoplasmic material [[15]], and chaperone-mediated autophagy the selective degradation of certain proteins in the lysosome [[16]]. Here, Sakai and Oku [[17]] discuss the process of microautophagy and present current findings on the control mechanisms of different forms of microautophagy. In addition to the early breakthrough discoveries in autophagy research mentioned above, the principle of selective autophagy with respect to the endoplasmic reticulum and peroxisomes was reported in both yeast and mammalian systems in the late 1970s [[18, 19]]. Since then, many different forms of selective autophagy have been reported [[20, 21]], and in our special issue here, Ryan and Rubinsztein [[22]] address selective autophagy of stress granules in the context of neurodegenerative diseases. Then, in 1993, the molecular era of autophagy research began with the isolation of S. cerevisiae mutants defective in autophagy, followed by the identification of the corresponding yeast ATG (autophagy related) genes [[2, 23, 24]]. The sophisticated molecular mechanisms of autophagosome biogenesis and their fusion with the vacuole with a focus on S. cerevisiae as a model organism are detailed in the graphical review by Shatz and Elazar [[25]], and the review by Kraft and Reggiori [[26]]. The elegant structural and functional relationship of the autophagic machinery is further dissected in the review by Noda [[27]]. The review by Tooze et al. [[28]] pays particular attention to new, profound molecular insights into the specific role of ATG8 in autophagosome biogenesis. This review article demonstrates the molecular detail with which current autophagy research provides important insights into the specific functions of the individual parts of the autophagy machinery. Moreover, a detailed insight into the regulation of the initiation complex for autophagosome biogenesis is provided by describing the metamorphosis of the HORMA domain proteins ATG13 and ATG101 in the review by Nguyen and Faesen [[29]]. With the identification and characterization of ATG genes in mammals, often highly conserved orthologues and paralogues of the yeast ATG genes, the physiological roles of autophagy and its enormous relevance for many human diseases, such as cancer and neurodegeneration, begun to be unraveled [[30-32]]. In fact, the discovery of human BECN1/Beclin 1 in 1999 as a haploinsufficient tumor suppressor [[30]] and essential component of the PI-3 kinase complex that produces PI3P during autophagy initiation has triggered tremendous enthusiasm to study autophagy due to its relevance to human cancer [[33]]. In this context, the human WIPI genes encoding PI3P-binding β-propellers that evolved from the ancestral yeast Atg18 [[34, 35]] have been identified in 2004 to be aberrantly expressed in human cancer and to fulfill important roles in autophagy [[36]]. Their functions as PI3P effectors in autophagy and consequences of human WIPI gene mutations causing rare forms of neurodegeneration, as found initially for WDR45/WIPI4 [[37, 38]], are summarized here by Proikas-Cezanne et al. [[39]]. The special role of autophagy in fighting more common forms of neurodegenerative diseases is highlighted by Ryan and Rubinsztein [[22]], a pioneer in unrevealing the functional connection between autophagy and neurodegeneration [[40]]. In the context of deciphering the role of autophagy in maintaining human health, it is further important to highlight that autophagy has a specific role in innate immunity [[41]], as discussed in the comprehensive review by Colombo and colleagues [[42]]. Particularly in recent years, the causal relationship between defective autophagy and human diseases has begun to be understood in molecular detail. However, despite major advances in understanding the mechanism and importance of autophagy for human health, key questions remain unanswered. How can autophagy be modulated therapeutically to treat various human diseases? Would it be advisable to enhance autophagy in advanced age to prevent neurodegeneration? And at the mechanistic level, how is phagophore closure and autophagosome maturation regulated? We can therefore remain curious to see what the next 30 years of autophagy research will bring. But one thing is for sure. It continues to be an exciting journey. Tassula Proikas-Cezanne is a professor (APL) for molecular biology and cell biology at the University of Tübingen. She identified the human WIPI genes and her interest focusses on the regulation of WIPI β-propellers and their roles in the process of autophagy in the context of cancer and neurodegeneration. Michael Thumm is a professor for biochemistry and molecular cell biology at the University of Göttingen. He identified key yeast ATG genes, and since then, his long-standing research interest lays on the molecular mechanism and the physiological function of macro- and microautophagy in the yeast S. cerevisiae.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.

Atg18, Atg21 and Hsv2 are homologous β-propeller proteins binding to PI3P and PI(3,5)P2. Atg18 is thought to organize lipid transferring protein complexes at contact sites of the growing autophagosome (phagophore) with both the ER and the vacuole. Atg21 is restricted to the vacuole phagophore contact, where it organizes part of the Atg8-lipidation machinery. The role of Hsv2 is less understood, it partly affects micronucleophagy. Atg18 is further involved in regulation of PI(3,5)P2 synthesis. Recently, a novel Atg18-retromer complex and its role in vacuole homeostasis and membrane fission was uncovered.

The yeast PROPPIN Atg18 folds as a β-propeller with two binding sites for phosphatidylinositol-3-phosphate (PtdIns3P) and PtdIns(3,5)P2 at its circumference. Membrane insertion of an amphipathic loop of Atg18 leads to membrane tubulation and fission. Atg18 has known functions at the PAS during macroautophagy, but the functional relevance of its endosomal and vacuolar pool is not well understood. Here we show in a proximity-dependent labeling approach and by co-immunoprecipitations that Atg18 interacts with Vps35, a central component of the retromer complex. The binding of Atg18 to Vps35 is competitive with the sorting nexin dimer Vps5 and Vps17. This suggests that Atg18 within the retromer can substitute for both the phosphoinositide binding and the membrane bending capabilities of these sorting nexins. Indeed, we found that Atg18-retromer is required for PtdIns(3,5)P2-dependent vacuolar fragmentation during hyperosmotic stress. The Atg18-retromer is further involved in the normal sorting of the integral membrane protein Atg9. However, PtdIns3P-dependent macroautophagy and the selective cytoplasm-to-vacuole targeting (Cvt) pathway are only partially affected by the Atg18-retromer. We expect that this is due to the plasticity of the different sorting pathways within the endovacuolar system.Abbreviations: BAR: bin/amphiphysin/Rvs; FOA: 5-fluoroorotic acid; PAS: phagophore assembly site; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology.

Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes.Abbreviations: DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.

Coupling of Atg8 to phosphatidylethanolamine is crucial for the expansion of the crescent-shaped phagophore during cargo engulfment. Atg21, a PtdIns3P-binding beta-propeller protein, scaffolds Atg8 and its E3-like complex Atg12-Atg5-Atg16 during lipidation. The crystal structure of Atg21, in complex with the Atg16 coiled-coil domain, showed its binding at the bottom side of the Atg21 beta-propeller. Our structure allowed detailed analyses of the complex formation of Atg21 with Atg16 and uncovered the orientation of the Atg16 coiled-coil domain with respect to the membrane. We further found that Atg21 was restricted to the phagophore edge, near the vacuole, known as the vacuole isolation membrane contact site (VICS). We identified a specialized vacuolar subdomain at the VICS, typical of organellar contact sites, where the membrane protein Vph1 was excluded, while Vac8 was concentrated. Furthermore, Vac8 was required for VICS formation. Our results support a specialized organellar contact involved in controlling phagophore elongation.

Nucleophagy, the selective subtype of autophagy that targets nuclear material for autophagic degradation, was not only shown to be a model system for the study of selective macroautophagy, but also for elucidating the role of the core autophagic machinery within microautophagy. Nucleophagy also emerged as a system associated with a variety of disease conditions including cancer, neurodegeneration and ageing. Nucleophagic processes are part of natural cell development, but also act as a response to various stress conditions. Upon releasing small portions of nuclear material, micronuclei, the autophagic machinery transfers these micronuclei to the vacuole for subsequent degradation. Despite sharing many cargos and requiring the core autophagic machinery, recent investigations revealed the aspects that set macro- and micronucleophagy apart. Central to the discrepancies found between macro- and micronucleophagy is the nucleus vacuole junction, a large membrane contact site formed between nucleus and vacuole. Exclusion of nuclear pore complexes from the junction and its exclusive degradation by micronucleophagy reveal compositional differences in cargo. Regarding their shared reliance on the core autophagic machinery, micronucleophagy does not involve normal autophagosome biogenesis observed for macronucleophagy, but instead maintains a unique role in overall microautophagy, with the autophagic machinery accumulating at the neck of budding vesicles.

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.

In Saccharomyces cerevisiae Atg8 coupled to phosphatidylethanolamine is a key component of autophagosome biogenesis. Atg21 binds via 2 sites at the circumference of its β-propeller to PtdIns3P at the phagophore assembly site (PAS). It recruits and arranges both Atg8 and Atg16, which is part of the E3-like ligase complex Atg12-Atg5-Atg16. Binding of Atg8 to Atg21 requires the FK-motif within the N-terminal-helical domain of Atg8 and D146 at the top of the Atg21 β-propeller. Atg16 binds via D101 and E102 within its coiled-coil domain to Atg21.

Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method.

PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding.