Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation.This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes. INTRODUCTIONAutophagy occurs in all eukaryotic cells (Reggiori and Klionsky, 2002). In yeast it has mainly been characterized as an adaptation to nutrient stress such as nitrogen limitation: during autophagy large portions of cytosolic and membraneous material are delivered to the lysosome (in yeast called vacuole) for degradation and recycling (Takeshige et al., 1992). This phenomenon enables cells to survive long periods of starvation. However, in higher eukaryotes autophagy also plays an important role in developmental changes (Levine and Klionsky, 2004), regulation of lifespan (Bergamini et al., 2003;Longo and Finch, 2003;Melendez et al., 2003;Vellai et al., 2003), cancer (Qu et al., 2003;Yue et al., 2003;Gozuacik and Kimchi, 2004), and in neurodegenerative disorders like Huntington's, Parkinson's, or Alzheimer's disease (Yuan et al., 2003). It is also part of the innate immune system assisting in eliminating intracellular pathogens after infection (Gutierrez et al., 2004;Nakagawa et al., 2004;Ogawa et al., 2005).Macroautophagy in yeast is defined as the uptake of cytosolic contents by fusion of double-layered vesicles (autophagosomes) with vacuoles (Baba et al., 1994). Autophagosomes originate from a preautophagosomal structure (PAS) in the vicinity of the vacuole and, during their formation, enwrap portions of cytosol. Fusion of the outer autophagosomal layer with the vacuolar membrane liberates autophagic bodies (single-layered intravacuolar vesicles) into the vacuolar lumen for degradation (Takeshige et al., 1992). Relevant actors of macroautophagy (Atg proteins; Klionsky et al., 2003) have been identified, mainly by genetic screens (Tsukada and Ohsumi, 1993;Thumm et al., 1994;Harding et al., 1995Harding et al., , 1996Titorenko et al., 1995) and have been studied intensively over the last decade.Little is known about microautophagy, a process consisting of a di...

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. At the tip of an autophagic tube vesicles (autophagic bodies) pinch off into thevacuolar lumen for degradation. Formation of autophagic tubes is topologically equivalent to other budding processes directed away from the cytosolic environment, e.g., the invagination of multivesicular endosomes, retroviral budding, piecemeal microautophagy of the nucleus and micropexophagy. This clearly distinguishes microautophagy from other membrane fission events following budding toward the cytosol. Such processes are implicated in transport between organelles like the plasma membrane, the endoplasmic reticulum (ER), and the Golgi. Over many years microautophagy only could be characterized microscopically. Recent studies provided the possibility to study the process in vitro and have identified the first molecules that are involved in microautophagy.

Microautophagy is the uptake of cytosolic compounds by direct invagination of the vacuolar/lysosomal membrane. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. Autophagic tubes are topologically equivalent to the invaginations at multivesicular endosomes. At the tip of an autophagic tube, vesicles (autophagic bodies) pinch off into the vacuolar lumen for degradation. In this study we have identified calmodulin (Cmd1p) as necessary for microautophagy. Temperaturesensitive mutants for Cmd1p displayed reduced frequencies of vacuolar tube formation and/or abnormal tube morphologies. Microautophagic vacuole invagination was sensitive to Cmd1p antagonists as well as to antibodies to Cmd1p. cmd1 mutants with substitutions in the Ca 2؉ -binding domains showed full invagination activity, and vacuolar membrane invagination was independent of the free Ca 2؉ concentration. Thus, rather than acting as a calcium-triggered switch, Cmd1p has a constitutive Ca 2؉ -independent role in the formation of autophagic tubes. Kinetic analysis indicates that calmodulin is required for autophagic tube formation rather than for the final scission of vesicles from the tip of the tube.In eukaryotic cells there are many trafficking pathways between organelles. Many of these transport processes, such as the endocytic pathway, autophagocyotosis, cytoplasm to vacuole targeting, piecemeal microautophagy of the nucleus, and biosynthetic delivery of hydrolases, end at the lysosome, the main compartment for storage and degradation (1-6). In yeast the lysosomal organelle is called a vacuole.Under conditions of nutrient restriction, cytosolic and membraneous material reaches the vacuole by means of autophagy (7). Macroautophagy in yeast is defined as the uptake of cytosolic elements by fusion of double membrane vesicles (autophagosomes) with vacuoles (8). These double-layered vesicles are produced from preautophagosomal structures and, during their formation, excise portions of cytosol and enwrap them. Fusion of the outer autophagosomal membrane with lysosomes produces single-layered intravacuolar vesicles (autophagic bodies) that are degraded (7). Macroautophagy has been studied intensively over the last decade, and many relevant components (Atg proteins) (9) have been identified, mainly by genetic screens (10 -14). In contrast, only little is known about a process consisting of a direct vacuolar invagination and budding of autophagic bodies into the vacuolar lumen, which we call microautophagy of soluble compounds (15-17). Besides microautophagy of soluble cytosolic components, also larger particles can be taken up by vacuoles, e.g. during piecemeal microautophagy of the nucleus (PMN) 2 (6), which transfers parts of the nucleus into vacuoles, and during micropexophagy (18 -23), which leads to the degradation of peroxisomes. Pexophagic vacuole invagination depends on Atg proteins (24 -27) and hence shares components with the macroautoph...