Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

LonB Protease Is a Novel Regulator of Carotenogenesis Controlling Degradation of Phytoene Synthase in <i>Haloferax volcanii</i>

The membrane protease LonB is an essential protein in the archaeon <i>Haloferax volcanii</i> and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a <i>H. volcanii</i> LonB mutant under reduced and physiological protease levels. LC–MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis

Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

<div><p>Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.</p></div

The absence of GBA2 affects cell migration.

<p><b>(A-B)</b> Wound-healing assay to analyze cell migration of dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). <b>(A)</b> Representative images at different time points after initiating the assay. Scale bars are indicated. <b>(B)</b> Analysis of cell migration. The rate of cell migration has been analyzed. Average data points for wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) for different time points are shown; n numbers and p values using One-Way ANOVA are indicated.</p

Lack of GBA2 results in accumulation of GlcCer.

<p><b>(A)</b> Quantitative analysis of neutral sphingolipids in adult testis. +/+: wild-type;-/-: GBA2 knockout. LCB: long-chain bases, Cer: ceramides, HexCer: hexosylceramides, Spm: sphingomyelins. <b>(B)</b> See (A) for P7 Sertoli cells. <b>(C)</b> See (A) for sperm. <b>(D)</b> Quantitative analysis of HexCer in adult testis. Lipids are classified according to their acyl chain-length. <b>(E)</b> See (D) for sperm. <b>(F)</b> See (D) for very long chain fatty acids. All data are presented as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p

Dermal fibroblasts from GBA2 knockout-mice also display cytoskeletal defects.

<p><b>(A)</b> GBA2 expression in dermal fibroblasts from adult mice. Total protein lysates were probed with a GBA2-specific antibody (2F8) on a Western blot. Heterologously expressed HA-tagged GBA2 was used as a positive control, calnexin (Clnx) as a loading control. <b>(B)</b> Accumulation of GlcCer in GBA2 knockout-fibroblasts. Thin layer chromatography (TLC) analyzing glycosphingolipids from wild-type (+/+) and GBA2 knockout-fibroblasts (-/-). Representative TLC analysis for neutral sphingolipids. GlcCer: glucosylceramide, LacCer: lactosylceramide, Spm: sphingomyelin. GlcCer levels were quantified by densitometry and are presented as mean ± S.D. The fold change in GlcCer levels in GBA2 knockout-fibroblasts was calculated. <b>(C)</b> Fluorescent labeling of the cytoskeleton in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were transfected with lifeact (green) to visualize F-actin and with EB3-cherry to visualize microtubules. Scale bars are indicated. <b>(D)</b> Fluorescent labeling of F-actin in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were seeded on CYTOO chips with micropatterns that are coated with fluorescently-labeled fibronectin (purple). F-actin was stained using Alexa Fluor Phalloidin 488 (green) and the DNA was stained with DAPI (blue). Scale bars are indicated. <b>(E)</b> Analysis of cytoskeletal structures. Cells were seeded on the crossbow shape. The number of cells containing filopodia or lamellipodia (left) and the average number of filopodia or lamellipodia per cell (right) were determined. <b>(F)</b> Gene expression-analysis. The mRNA expression level of <i>Cdc42</i>, <i>Rac1</i>, and <i>Rho</i> was analyzed by qRT-PCR. <b>(G)</b> Protein expression-analysis. Total protein lysates were probed with a GBA2- (2F8), a Cdc42-, and a Rac1-specific antibody on a Western blot. Calnexin (Clnx) was used as a loading control. <b>(H)</b> Quantification of protein expression based on (G). <b>(I)</b> Quantification of actin turnover in dermal fibroblasts. Expression levels of G- and F-actin in wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were determined using Western blot-analysis. Ratio of F-actin/G-actin for wild-type and GBA2 knockout-fibroblasts is expressed relative to the control. <b>(J)</b> See (I) for testis. <b>(K-M)</b> Analysis of microtubule dynamics in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). <b>(K)</b> Expression of EB3-cherry in dermal fibroblasts. Cells were transfected with EB3-cherry and microtubule dynamics were analyzed. Representative tracks of growing microtubule plus-ends are indicated with white lines. <b>(L)</b> Microtubule growth rate. Wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were transfected with EB3-cherry and the growth rate of growing plus-ends was analyzed. Per genotype, n = 3 animals with a minimum of 7 cells and 10 tracks per cell were analyzed. Data are presented as mean ± S.D. <b>(M)</b> see (L) for microtubule persistence. For all bar graphs, data are shown as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p

GBA2 is expressed in Sertoli cells.

<p><b>(A)</b> GBA2 expression in testis. Total protein lysates were probed with a GBA2-specific antibody (2F8) on a Western blot. Beta-tubulin was used as a loading control. +/+: wild-type;-/-: GBA2 knockout. <b>(B)</b> GBA2 expression in mouse sperm. Total protein lysates were probed with a GBA2-specific antibody (2F8) on a Western blot. Heterologously expressed HA-tagged GBA2 was used as a positive control, beta-tubulin as a loading control. <b>(C)</b> Immunohistochemical analysis of GBA2 in testis. Testis cross-sections were labeled with a GBA2-specific antibody (pcGBA2, red) and an anti-beta tubulin III antibody (green) as a marker for Sertoli cells. DAPI was used to label the DNA. Scale bars are indicated. <b>(D)</b> GBA2 expression in P7 Sertoli cells. See (B). <b>(E-G)</b> GBA2 expression during development. <b>(E)</b> See (C) for testis cross-sections from P7, P21, and adult wild-type mice. <b>(F)</b> Representative Western blot using protein lysates from wild-type testis at P7, P21, and adult mice (ad). Heterologously expressed HA-tagged GBA2 has been used as a positive control and beta-tubulin III as a Sertoli-cell marker (TubIII). Protein lysates from wild-type (+/+) and GBA2 knockout-brain (-/-) are shown as controls. <b>(G)</b> Quantification of GBA2 protein expression. Expression levels of GBA2 have been normalized to beta-tubulin III. Data are presented as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated. <b>(H)</b> GBA2 activity during sperm development. Beta-glucosidase activity was measured in protein lysates from wild-type testis at pH 6 using 1.67 mM of the artificial substrate 4-methylumbelliferyl-beta-D-glucopyranoside. Data are presented as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p