Roswitha Krick

Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8

Cdc48/p97/VCP plays a ubiquitin-independent role during autophagosome formation in S. cerevisiae.

Turnover of organelles by autophagy in yeast

Efficient detection and removal of superfluous or damaged organelles are crucial to maintain cellular homeostasis and to assure cell survival. Growing evidence shows that organelles or parts of them can be removed by selective subtypes of otherwise unselective macroautophagy and microautophagy. This requires both the adaptation of the core autophagic machinery and sophisticated mechanisms to recognize organelles destined for turnover. We review the current knowledge on autophagic removal of peroxisomes, mitochondria, ER and parts of the nucleus with an emphasis on yeasts as a model eukaryote.

Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family

β-propellers that bind polyphosphoinositides (PROPPINs), a eukaryotic WD-40 motif-containing protein family, bind via their predicted β-propeller fold the polyphosphoinositides PtdIns3P and PtdIns(3,5)P2 using a conserved FRRG motif. PROPPINs play a key role in macroautophagy in addition to other functions. We present the 3.0-Å crystal structure of Kluyveromyces lactis Hsv2, which shares significant sequence homologies with its three Saccharomyces cerevisiae homologs Atg18, Atg21, and Hsv2. It adopts a seven-bladed β-propeller fold with a rare nonvelcro propeller closure. Remarkably, in the crystal structure, the two arginines of the FRRG motif are part of two distinct basic pockets formed by a set of highly conserved residues. In comprehensive in vivo and in vitro studies of ScAtg18 and ScHsv2, we define within the two pockets a set of conserved residues essential for normal membrane association, phosphoinositide binding, and biological activities. Our experiments show that PROPPINs contain two individual phosphoinositide binding sites. Based on docking studies, we propose a model for phosphoinositide binding of PROPPINs.

The relevance of the phosphatidylinositolphosphat‐binding motif FRRGT of Atg18 and Atg21 for the Cvt pathway and autophagy

Atg18 and Atg21 are homologous S. cerevisiae autophagy proteins. Atg18 is essential for biogenesis of Cvt vesicles and autophagosomes, while Atg21 is only essential for Cvt vesicle formation. We found that mutated Atg18‐(FTTGT), which lost almost completely its binding to PtdIns3P and PtdIns(3,5)P2, is non‐functional during the Cvt pathway but active during autophagy and pexophagy. Since the Cvt pathway does not depend on PtdIns(3,5)P2, we conclude that the Cvt pathway requires binding of Atg18 to PtdIns3P. Mutated Atg21‐(FTTGT) is inactive during the Cvt pathway but showed only partly reduced binding to PtdIns‐phosphates, suggesting further lipid binding domains in Atg21. GFP‐Atg18‐(FTTGT) and Atg21‐(FTTGT)‐GFP are released from vacuolar punctae to the cytosol.

Dissecting the localization and function of Atg18, Atg21 and Ygr223c.

Atg18p and Atg21p are two highly homologous yeast autophagy proteins. Atg18p functions in both autophagy and the selective Cvt-pathway, while the function of Atg21p is restricted to the Cvt-pathway. The yeast genome encodes with Ygr223cp (Hsv2p) a third member of this protein family. So far no function has been assigned to Ygr223cp. By colocalization with the endosomal marker Snf7-RFP and an RFP-tagged FYVE domain, we here identify the localization of a pool of Atg18p, Atg21p and Ygr223cp at endosomes. Endosomal recruitment of all three proteins depends on PtdIns3P generated by the Vps34-complex II containing Vps38p, but not on the function of the Vps34-complex I. Since only the Vps34-complex I is essential for autophagy, we expect that at endosomes Atg18p, Atg21p and Ygr223cp have a function distinct from autophagy. Some Vps Class D mutants involved in Golgi-to-endosome transport are required for the endosomal recruitment of GFP-Atg18p, -Atg21p and –Ygr223cp. These include the Qa-SNARE Pep12p, its SM protein Vps45p, the Rab GTPase Vps21p and the Rab effector Vac1p. Deletion of ATG18, ATG21 and YGR223c, alone or simultaneously has no obvious function on the MVB-pathway and CPY-sorting. However, overexpression of ATG21 leads to CPY secretion. We further show, to our knowledge for the first time that Ygr223cp affects an autophagic process, namely micronucleophagy.

GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis.

Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.

Quantification of nonselective bulk autophagy in S. cerevisiae using Pgk1-GFP

Rapid estimation of the macroautophagic rate has become of great importance over the last few years. A variety of methods to follow autophagy were established both in S. cerevisiae and the mammalian system. In yeast, measuring the breakdown of GFP-Atg8, and in mammalian cells counting the increase of LC3 puncta, have become the most commonly used assays to quantify autophagy. Here, we provide degradation of Pgk1-GFP followed in immunoblots as a new convenient tool to quantify nonselective bulk autophagy in yeast.

Measuring piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae.

Piecemeal microautophagy of the nucleus (PMN) selectively removes and degrades small fragments of the Saccharomyces cerevisiae nucleus. Inter-organelle contact sites called nucleus-vacuole (NV) junctions determine the selectivity of PMN by establishing a platform for the biogenesis of PMN blebs and vesicles. PMN structures can be observed by fluorescence microscopy using GFP-tagged reporters; however, this approach is best supported with quantitative immunoblot assays of PMN-specific cargo degradation. Together, these assays should facilitate the further study of this fascinating but poorly understood autophagic process in different genetic backgrounds, physiological states, and environmental conditions.

Uth1 is a mitochondrial inner membrane protein dispensable for post-log-phase and rapamycin-induced mitophagy

Mitochondria are turned over by an autophagic process termed mitophagy. This process is considered to remove damaged, superfluous and aged organelles. However, little is known about how defective organelles are recognized, what types of damage induce turnover, and whether an identical set of factors contributes to degradation under different conditions. Here we systematically compared the mitophagy rate and requirement for mitophagy‐specific proteins during post‐log‐phase and rapamycin‐induced mitophagy. To specifically assess mitophagy of damaged mitochondria, we analyzed cells accumulating proteins prone to degradation due to lack of the mitochondrial AAA‐protease Yme1. While autophagy 32 (Atg32) was required under all tested conditions, the function of Atg33 could be partially bypassed in post‐log‐phase and rapamycin‐induced mitophagy. Unexpectedly, we found that Uth1 was dispensable for mitophagy. A re‐evaluation of its mitochondrial localization revealed that Uth1 is a protein of the inner mitochondrial membrane that is targeted by a cleavable N‐terminal pre‐sequence. In agreement with our functional analyses, this finding excludes a role of Uth1 as a mitochondrial surface receptor.

PI3P binding by Atg21 organises Atg8 lipidation

Autophagosome biogenesis requires two ubiquitin‐like conjugation systems. One couples ubiquitin‐like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin‐like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P‐containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 β‐propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled‐coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6‐motif in the N‐terminal helical domain of Atg8, but not its AIM‐binding site. Accordingly, the Atg8 AIM‐binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P‐dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.