The yeast cell wall protein Pry3 inhibits mating through highly conserved residues within the CAP domain.

Members of the CAP/SCP/TAPS superfamily have been implicated in many different physiological processes, including pathogen defense, sperm maturation and fertilization. The mode of action of this class of proteins, however, remains poorly understood. The genome of Saccharomyces cerevisiae encodes three CAP superfamily members, Pry1-3. We have previously shown that Pry1 function is required for the secretion of sterols and fatty acids. Here, we analyze the function of Pry3, a GPI-anchored cell wall protein. Overexpression of Pry3 results in strong reduction of mating efficiency, providing for a cell-based readout for CAP protein function. Mating inhibition is a conserved function of the CAP domain and depends on highly conserved surface exposed residues that form part of a putative catalytic metal-ion binding site. Pry3 displays polarized cell surface localization adjacent to bud scars, but is absent from mating projections. When overexpressed, however, the protein leaks onto mating projections, suggesting that mating inhibition is due to mislocalization of the protein. Trapping of the CAP domain within the cell wall through a GPI-anchored nanobody results in a dose-dependent inhibition of mating, suggesting that a membrane proximal CAP domain inhibits a key step in the mating reaction, which is possibly related to the function of CAP domain proteins in mammalian fertilization.This article has an associated First Person interview with the first author of the paper.

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).

Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles.

Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro. Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion pathways remain uncharacterized. In this article, we show that B. fragilis and B. thetaiotaomicron preferentially pack a large number of hydrolases in outer membrane vesicles (OMV). Most of these hydrolases are acidic and were detected exclusively in OMV. This suggests the presence of a molecular mechanism in Bacteroides responsible for the selection of OMV proteins based on their charge. We propose that OMV contribute to the establishment and balance of the gut microbiota.

Ubp15p, a ubiquitin hydrolase associated with the peroxisomal export machinery.

Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.

The putative Saccharomyces cerevisiae hydrolase Ldh1p is localized to lipid droplets.

Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae. Ldh1p (systematic name, Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the α/ß-hydrolase family and shares some features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signal type1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storage compartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, and targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.

Involvement of the Saccharomyces cerevisiae hydrolase Ldh1p in lipid homeostasis.

Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.

Lpx1p is a peroxisomal lipase required for normal peroxisome morphology.

Lpx1p (systematic name: Yor084wp) is a peroxisomal protein from Saccharomyces cerevisiae with a peroxisomal targeting signal type 1 (PTS1) and a lipase motif. Using mass spectrometry, we have identified Lpx1p as present in peroxisomes, and show that Lpx1p import is dependent on the PTS1 receptor Pex5p. We provide evidence that Lpx1p is piggyback-transported into peroxisomes. We have expressed the Lpx1p protein in Escherichia coli, and show that the enzyme exerts acyl hydrolase and phospholipase A activity in vitro. However, the protein is not required for wild-type-like steady-state function of peroxisomes, which might be indicative of a metabolic rather than a biogenetic role. Interestingly, peroxisomes in deletion mutants of LPX1 have an aberrant morphology characterized by intraperoxisomal vesicles or invaginations.

The AAA peroxins Pex1p and Pex6p function as dislocases for the ubiquitinated peroxisomal import receptor Pex5p.

The discovery of the peroxisomal ATPase Pex1p triggered the beginning of the research on AAA (ATPase associated with various cellular activities) proteins and the genetic dissection of peroxisome biogenesis. Peroxisomes are virtually ubiquitous organelles, which are connected to diverse cellular functions. The highly diverse and adaptive character of peroxisomes is accomplished by modulation of their enzyme content, which is mediated by dynamically operating protein-import machineries. The import of matrix proteins into the peroxisomal lumen has been described as the ATP-consuming step, but the corresponding reaction, as well as the ATPase responsible, had been obscure for nearly 15 years. Recent work using yeast and human fibroblast cells has identified the peroxisomal AAA proteins Pex1p and Pex6p as mechano-enzymes and core components of a complex which dislocates the cycling import receptor Pex5p from the peroxisomal membrane back to the cytosol. This AAA-mediated process is regulated by the ubiquitination status of the receptor. Pex4p [Ubc10p (ubiquitin-conjugating enzyme 10)]-catalysed mono-ubiquitination of Pex5p primes the receptor for recycling, thereby enabling further rounds of matrix protein import, whereas Ubc4p-catalysed polyubiquitination targets Pex5p to proteasomal degradation.