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)

Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis

A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum–derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes. We tested these models by reexamining the role of Pex1/Pex6 and dynamin-related proteins in peroxisome biogenesis. We found that induced depletion of Pex1 blocks the import of matrix proteins but does not affect membrane protein delivery to peroxisomes; markers for the previously reported distinct vesicles colocalize in pex1 and pex6 cells; peroxisomes undergo continued growth if ission is blocked. Our data are compatible with the established primary role of the Pex1/Pex6 complex in matrix protein import and show that peroxisomes in Saccharomyces cerevisiae multiply mainly by growth and division

Characterization of peroxisome- and lipid droplet-related proteins of Saccharomyces cerevisiae\textit {Saccharomyces cerevisiae}

Es konnte gezeigt werden, dass Lpx1p in Peroxisomen lokalisiert ist, wo hingegen Ldh1p überwiegend zu Lipid-Droplets dirigiert wird. Während gezeigt werden konnte, das es sich bei Lpx1p um ein peroxisomales Enzym handelt, wurde anhand subzellulärer Lokalisationsstudien für Ldh1p eine überwiegende Lokalisation an Lipid-Droplets dargelegt. Für Lpx1p und Ldh1p konnte $\textit {in vitro}$ mittels rekombinanter Proteine eine Triacylglycerol-Lipase wie auch-hydrolase Aktivität belegt werden. Lpx1p ist am peroxisomalen Stoffwechsel beteiligt. Basierend auf den Daten wird eine Funktion von Ldh1p in der Aufrechterhaltung der Lipid-Homeostase in der Hefe durch die Regulation des Spiegels an Phospholipiden wie auch nicht-polaren Lipiden diskutiert. In der vorliegenden Arbeit konnte gezeigt werden, dass der AAA-Komplex sowohl die Pex5p-Dislokaseaktivität wie auch eine deubiquitinilierende Aktivität beinhaltet.It was shown that Lpx1p is present in the peroxisome but Ldh1p is predominantly localized to lipid droplets. While Lpx1p was shown to be a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets. Triacylglycerol lipase as well as hydrolase activities were shown for both recombinant proteins Lpx1p and Ldh1p $\textit {in vitro}$. It was shown that the Lpx1p 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. It was clearly shown that peroxisomes in $\Delta$$\it {lpx1}$ mutants have an aberrant morphology characterized by intraperoxisomal vesicles or invaginations. Ldh1p is not required for the function and biogenesis of peroxisomes. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. It was shown in this work that the AAA-complex contains Pex5p dislocase as well as deubiquitinating activity

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

Molecular mass and scores of XLs found by pLink for Pma1p and BSA.

<p>Distributions of scores and calculated molecular masses of XLs generated by pLink by scanning through all mgf files from BS3 experiments (<b>A-D</b>) or EDC experiments (<b>E-H</b>) for Pma1p and BSA, a protein that is not present in the sample, are plotted as calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s007" target="_blank">S7 Table</a>. The cut off lines used to filter all pLink XLs (stippled red lines) indicate a minimal molecular mass of XL of 1’500 Da and scores of ≤ 7.5 x 10⁻⁴ for BS3-XLs, or ≤ 4 x 10⁻⁴ for EDC-XLs.</p

Frequency of XLs found by pLink for a given protein is correlated with its copy number.

<p>The number of XLs for 18 arbitrary chosen MSPs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s004" target="_blank">S4 Table</a> is plotted as a function of the copy number of the respective MSP (<b>A</b>) or its amino acid length (<b>B</b>). In panel <b>C</b>, the number of BS3- and EDC-generated XLs for these 18 proteins are plotted separately, as a function of copy number. In panel <b>D</b>, the distance in the primary sequence between crosslinked amino acids in 160 XLs found in 27 proteins of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.s014" target="_blank">S14 Table</a> are plotted.</p

BS3 and EDC crosslinks mapped onto the structural models of Pma1p, Por1p and Sec61p.

<p>Structures of Pma1p (<b>A, B</b>), Por1p (<b>C-F</b>), and Sec61p (<b>G, H</b>) were homology modeled by HHPRED [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref015" target="_blank">15</a>] using plasma membrane H⁺-ATPase from <i>Neurospora crassa</i> (1mhs_A) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref016" target="_blank">16</a>], the voltage-dependent anion channel VDAC1 from <i>Mus musculus</i> (4c69_X) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref017" target="_blank">17</a>], and Sec61 from <i>Canis lupus/Bos Taurus</i> (3jc2_1) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186840#pone.0186840.ref018" target="_blank">18</a>] as template. Structural models were visualized by PyMOL and the position of crosslinks connecting the Cα atoms of amino acids were added manually based on the experimental data from pLink.</p