Vacuolar hydrolysis and efflux: current knowledge and unanswered questions.

Hydrolysis within the vacuole in yeast and the lysosome in mammals is required for the degradation and recycling of a multitude of substrates, many of which are delivered to the vacuole/lysosome by autophagy. In humans, defects in lysosomal hydrolysis and efflux can have devastating consequences, and contribute to a class of diseases referred to as lysosomal storage disorders. Despite the importance of these processes, many of the proteins and regulatory mechanisms involved in hydrolysis and efflux are poorly understood. In this review, we describe our current knowledge of the vacuolar/lysosomal degradation and efflux of a vast array of substrates, focusing primarily on what is known in the yeast Saccharomyces cerevisiae. We also highlight many unanswered questions, the answers to which may lead to new advances in the treatment of lysosomal storage disorders. Abbreviations: Ams1: α-mannosidase; Ape1: aminopeptidase I; Ape3: aminopeptidase Y; Ape4: aspartyl aminopeptidase; Atg: autophagy related; Cps1: carboxypeptidase S; CTNS: cystinosin, lysosomal cystine transporter; CTSA: cathepsin A; CTSD: cathepsin D; Cvt: cytoplasm-to-vacuole targeting; Dap2: dipeptidyl aminopeptidase B; GS-bimane: glutathione-S-bimane; GSH: glutathione; LDs: lipid droplets; MVB: multivesicular body; PAS: phagophore assembly site; Pep4: proteinase A; PolyP: polyphosphate; Prb1: proteinase B; Prc1: carboxypeptidase Y; V-ATPase: vacuolar-type proton-translocating ATPase; VTC: vacuolar transporter chaperone.

A newly characterized vacuolar serine carboxypeptidase, Atg42/Ybr139w, is required for normal vacuole function and the terminal steps of autophagy in the yeast Saccharomyces cerevisiae.

Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar serine carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.

An overview of autophagy: morphology, mechanism, and regulation.

SIGNIFICANCE: Autophagy is a highly conserved eukaryotic cellular recycling process. Through the degradation of cytoplasmic organelles, proteins, and macromolecules, and the recycling of the breakdown products, autophagy plays important roles in cell survival and maintenance. Accordingly, dysfunction of this process contributes to the pathologies of many human diseases. RECENT ADVANCES: Extensive research is currently being done to better understand the process of autophagy. In this review, we describe current knowledge of the morphology, molecular mechanism, and regulation of mammalian autophagy. CRITICAL ISSUES: At the mechanistic and regulatory levels, there are still many unanswered questions and points of confusion that have yet to be resolved. FUTURE DIRECTIONS: Through further research, a more complete and accurate picture of the molecular mechanism and regulation of autophagy will not only strengthen our understanding of this significant cellular process, but will aid in the development of new treatments for human diseases in which autophagy is not functioning properly.

Tannic acid inhibits Staphylococcus aureus surface colonization in an IsaA-dependent manner.

Staphylococcus aureus is a human commensal and pathogen that is capable of forming biofilms on a variety of host tissues and implanted medical devices. Biofilm-associated infections resist antimicrobial chemotherapy and attack from the host immune system, making these infections particularly difficult to treat. In order to gain insight into environmental conditions that influence S. aureus biofilm development, we screened a library of small molecules for the ability to inhibit S. aureus biofilm formation. This led to the finding that the polyphenolic compound tannic acid inhibits S. aureus biofilm formation in multiple biofilm models without inhibiting bacterial growth. We present evidence that tannic acid inhibits S. aureus biofilm formation via a mechanism dependent upon the putative transglycosylase IsaA. Tannic acid did not inhibit biofilm formation of an isaA mutant. Overexpression of wild-type IsaA inhibited biofilm formation, whereas overexpression of a catalytically dead IsaA had no effect. Tannin-containing drinks like tea have been found to reduce methicillin-resistant S. aureus nasal colonization. We found that black tea inhibited S. aureus biofilm development and that an isaA mutant resisted this inhibition. Antibiofilm activity was eliminated from tea when milk was added to precipitate the tannic acid. Finally, we developed a rodent model for S. aureus throat colonization and found that tea consumption reduced S. aureus throat colonization via an isaA-dependent mechanism. These findings provide insight into a molecular mechanism by which commonly consumed polyphenolic compounds, such as tannins, influence S. aureus surface colonization.

An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring.

ATP-binding cassette transporters are ubiquitous membrane protein complexes that move substrates across membranes. They do so using ATP-induced conformational changes in their nucleotide-binding domains to alter the conformation of the transport cavity formed by their transmembrane domains. In Escherichia coli, an ATP-binding cassette transporter-like complex composed of FtsE (nucleotide-binding domain) and FtsX (transmembrane domain) has long been known to be important for cytokinesis, but its role in the process has remained mysterious. Here we identify FtsEX as a regulator of cell-wall hydrolysis at the division site. Cell-wall material synthesized by the division machinery is shared initially by daughter cells and must be split by hydrolytic enzymes called "amidases" to drive daughter-cell separation. We recently showed that the amidases require activation at the cytokinetic ring by proteins with LytM domains, of which EnvC is the most critical. In this report, we demonstrate that FtsEX directly recruits EnvC to the septum via an interaction between EnvC and a periplasmic loop of FtsX. Importantly, we also show that FtsEX variants predicted to be ATPase defective still recruit EnvC to the septum but fail to promote cell separation. Our results thus suggest that amidase activation via EnvC in the periplasm is regulated by conformational changes in the FtsEX complex mediated by ATP hydrolysis in the cytoplasm. Since FtsE has been reported to interact with the tubulin-like FtsZ protein, our model provides a potential mechanism for coupling amidase activity with the contraction of the FtsZ cytoskeletal ring.

Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis.

During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC(-) defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.