The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy, Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole, As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis, This is the first characterization of a putative lipase implicated in vacuolar function in yeast.

The vacuole/lysosome performs a central role in degradation. Proteins and organelles are transported to the vacuole by selective and non-selective pathways. Transport to the vacuole by autophagy is the primary mode for degradation of cytoplasmic constituents under starvation conditions. Autophagy overlaps mechanistically and genetically with a biosynthetic pathway termed Cvt (Cytoplasm-to-vacuole targeting) that operates under vegetative conditions to transport the resident vacuolar hydrolase aminopeptidase I (API). API import has been dissected to reveal the action of a novel mechanism that transports cargo within double-membrane vesicles. Recent work has uncovered molecular components involved in autophagy and the Cvt pathway.

Several protein-targeting fields have recently converged in their observations of what once was thought to be a rare phenomenon: the transport of folded and oligomerized proteins across membranes. Three of the newly characterized pathways that are known to accommodate folded substrates are the peroxisomal targeting machinery for matrix proteins, the twin-arginine translocation (Tat) of bacteria and the related Delta pH-dependent pathway of plant plastids, and the cytoplasm-to-vacuole targeting (Cvt) pathway in Saccharomyces cerevisiae. Current work strives to understand the molecular mechanisms that accomplish transport of folded substrates. The aim of this commentary is to highlight our knowledge of transport mechanisms, point out areas for future research and address how paradigms of classical protein translocation have shaped current views.

We investigated the operation of a posttranslational protein translocation pathway to determine whether ions are excluded from the translocase during protein transport, The membrane capacitance during protein translocation across chloroplast thylakoid membranes was monitored via electric-field-indicating carotenoid electrochromic bandshift measurements. Evidence is presented that shows that the membrane ion conductance is not increased during the complete cycle of binding, transport, and substrate release by the Delta pH-dependent translocase; i.e., the membrane remains ion-tight during protein translocation, We further demonstrate that a synthetic targeting peptide that directs proteins across this membrane does not gate translocation pores, We conclude that protein transport across the thylakoid membrane does not compromise its ability to maintain ion gradients and is, thus, unlikely to affect its functions in energy transduction.

Recent work has clearly demonstrated a direct correlation between the amount of trehalose present in the yeast Saccharomyces cerevisiae and its ability to tolerate dehydration, but has failed to elucidate the specific role played by trehalose. By using Fourier transform infrared spectroscopy we measured the transition temperature of phospholipids in both intact S. cerevisiae and isolated plasma membranes dried in the presence and absence of trehalose. Our results show that trehalose lowers the temperature of the dry gel to liquid crystal phase transition in yeast from around 60 degrees C to about 40 degrees C, thus allowing yeast rehydrated above 40 degrees C to avoid the damaging effects of passing through a phase transition. These results explain both the need for trehalose and the observation that yeast must be rehydrated with warm water if they are to remain viable. Only when trehalose is present is the dry transition within a physiologically tolerable range and only when the cells are rehydrated above 40 degrees C will they avoid passing through a phase transition.