Journal: Nature Communications

Article Title: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

doi: 10.1038/s41467-025-63125-7

Figure Lengend Snippet: a Schematic overview of the genetic manipulations for auxin-inducible degradation of Sac1. Sac1 is endogenously tagged with a miniIAA7-3xFlag tag and the auxin receptor At AFB2(F74A) mutant is exogenously expressed in cells. After addition of an IAA analogue, miniIAA7-3xFlag-Sac1 is recruited to the AtAFB2(F74A)-Cullin E3 ligase complex, resulting in polyubiquitination and proteasomal degradation of Sac1. b Western blot analysis of Sac1-degron A431 cell lysates after IAA addition for 0 to 4 h in A431 cells, blotted for miniIAA7 to detect Sac1. Normalized relative intensity of each band is marked under the blot. c Bar graph showing total PI(4)P levels ±S.E.M. (pmol/x10 6 cells) of Sac1-degron A431 cells treated with DMSO for 2 h (0) or IAA for 0.5, 1 or 2 h as measured by PRMC-MS. n = 3 biological replicates. One-way ANOVA with Dunnett’s multiple comparisons test. n.s. p = 0.7464, * p = 0.0167, **** p < 0.0001. d Representative confocal images of Sac1-degron A431 cells treated with DMSO (4 h) or IAA for 2 or 4 h and stained for GM130 ( cis- Golgi) or TGN46 ( trans- Golgi). e Bar graph depicting the percentage (mean ±S.E.M) of cells with intact, partially fragmented, or fully fragmented Golgi. n = 3 independent experiments. f Western blot analysis of Sac1-degron A431 cells treated with DMSO or IAA for 4 h and blotted for TGN46, Golgin-97, GM130 and GRASP55. g Representative TEM images of Sac1-degron A431 cells treated with DMSO or IAA for 2 h. The yellow arrows indicate the Golgi stacks.

Article Snippet: Plasmids harboring wild-type Sac1 (Sac1 wt ) and a phosphatase-dead mutant Sac1 (C389S, Sac1 mut ) were obtained from Addgene (#108127 and #108128, respectively).

Techniques: Mutagenesis, Western Blot, Staining

Journal: Nature Communications

Article Title: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

doi: 10.1038/s41467-025-63125-7

Figure Lengend Snippet: a Top terms in gene ontology enrichment analysis of the downregulated proteins in Sac1-degron A431 cells treated with IAA for 3 h compared to DMSO control (-Log 10 False Discovery Rate (FDR) with a fold change threshold of ≥ 1.30 or ≤ 0.77 and an adjusted p-value of ≤ 0.05). b Schematic overview illustrating the changes in protein levels of glycosylation enzymes in Sac1-degron A431 cells after 3 h IAA treatment. The Log 2 fold change (Log 2 FC) of individual enzyme is presented using a color gradient from magenta (−2) to green ( + 2). Glycosylation starts in the ER, where precursor glycan chains are added to the protein. The glycans are modified sequentially by glycosidases and glycosyltransferases along the ER and Golgi stacks. c Western blot analysis of TGN46 and B4GALT1 in Sac1-degron A431 cells treated with IAA for 0 to 4 h. d Bar graph showing normalized intensity of B4GALT1 and TGN46 (mean ±S.E.M) quantified from ( c ). n = 3 independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test. TGN46: n.s. p = 0.9964, p = 0.5307, *** p = 0.0004, p = 0.0001. B4GALT1: n.s. p = 0.8336, p = 0.9872, p = 0.2245, ** p = 0.0006. e Western blot analysis of lysates of HEK293A Sac1-degron cells transfected with pcDNA3.1 (empty vector), EGFP-Sac1-WT (Sac1 wt ) or EGFP-Sac1-C389S (Sac1 mut ), treated for 6 h with DMSO or IAA and blotted for B4GALT1 and GFP (Sac1). f Graph depicts the B4GALT1 degradation efficiency (mean ±S.E.M) in Sac1-degron HEK293A cells treated for 6 h with IAA and rescued with Sac1 wt or Sac1 mut . n = 5 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. p = 0.6379, ****p < 0.0001.

Article Snippet: Plasmids harboring wild-type Sac1 (Sac1 wt ) and a phosphatase-dead mutant Sac1 (C389S, Sac1 mut ) were obtained from Addgene (#108127 and #108128, respectively).

Techniques: Control, Glycoproteomics, Modification, Western Blot, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

doi: 10.1038/s41467-025-63125-7

Figure Lengend Snippet: a Violin plot showing the distribution of TGN pH values of Sac1-degron A431 cells transiently transfected with GalT-RpHLuorin2 and treated with DMSO or IAA for 0 to 3 h. n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. p > 0.9999, p = 0.9848, * p = 0.0480, **** p < 0.0001. b Violin plot depicting the distribution of TGN pH values of Sac1-degron A431 cells treated for 3 h with DMSO or IAA with or without 50 µM EN6. n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test. n.s. p = 0.4775, ** p = 0.0039, **** p < 0.0001. c Western blot analysis of Sac1-degron A431 cells treated for 4 h with DMSO or IAA with or without 50 µM EN6. Immunoblotted for TGN46, B4GALT1 and miniIAA7 (Sac1). d Bar graph showing average normalized immunoblot intensities from ( c ). n = 4 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. TGN46: *** p = 0.0004, p = 0.0002, **** p < 0.0001. B4GALT1: * p = 0.0127, ** p = 0.0022, p = 0.0026. miniIAA7: *** p = 0.0001, **** p < 0.0001. n.s. p = 0.7763. e Representative confocal images of Sac1-degron A431 cells treated for 4 h with DMSO or IAA with or without 50 µM EN6. Stained for DAPI, Golgin-97 and TGN46. f Violin plot depicts the distribution of the TGN46 intensity in the Golgi area calculated from ( e ). n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. p = 0.6182, **** p < 0.0001. g Cartoon illustrating the reversible assembly of the V 0 and V 1 regions of the V-ATPase complex. Upon assembly, ATP hydrolysis of the V 1 region drives the proton pumping ability of the V 0 region. h Representative fluorescence micrographs of Sac1-degron A431 cells treated with DMSO for 4 h or IAA for 2 or 4 h and stained for ATP6V 0 a2 or ATP6V 1 A. i Violin plot showing the distribution of the Pearson’s correlation coefficient between ATP6V 0 a2 and ATP6V 1 A from ( h ). Representative data from one of three different experiments. One-way ANOVA with Dunnett’s multiple comparisons test. **** p < 0.0001. Representative confocal images of Sac1-degron A549 cells treated with DMSO or IAA for 2 h and stained for ATP6V 1 A and Golgin-97 ( j ) or ATP6V 1 A and LAMP2 ( l ). k , m Violin plot showing the Pearson’s correlation coefficient between ATP6V 1 A and Golgin-97 ( j ) and ATP6V 1 A and LAMP2 ( l ). n = 3 independent experiments. Two-tailed unpaired Student’s t-test. **** p < 0.0001, n.s. p = 0.1287.

Article Snippet: Plasmids harboring wild-type Sac1 (Sac1 wt ) and a phosphatase-dead mutant Sac1 (C389S, Sac1 mut ) were obtained from Addgene (#108127 and #108128, respectively).

Techniques: Transfection, Comparison, Western Blot, Staining, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

doi: 10.1038/s41467-025-63125-7

Figure Lengend Snippet: a Representative confocal images of Sac1-degron A431 cells treated with IAA or DMSO for 1 h and stained for PI(4)P and TGN46. b Violin plot showing the average PI(4)P intensity in the Golgi area (TGN46). Representative data from one of three independent experiments. Two-tailed unpaired Student’s t-test. **** p < 0.0001. c Representative confocal images of Sac1-degron A431 cell treated with IAA or DMSO for 2 h and stained with Filipin and Golgin-97. d Violin plot showing the normalized filipin intensity in the Golgi area (Golgin-97). Data from 3 independent experiments. Two-tailed Mann-Whitney U test. **** p < 0.0001. e Western blot analysis of sucrose gradient fractionations (Sucrose concentration from 0.3 M (fraction 1) to 2.1 M (loading fractions 9 – 12)) of A549 Sac1-degron cells treated for 2 h with DMSO, IAA, IAA with cholesterol, or IAA with PI4K inhibitors (NC03 and PIK93) and blotted for ATP6V 0 a2. f Line graph depicting the average percentage (mean±S.E.M.) of floating ATP6V 0 a2 distributed between fraction 1 to 7 of A549 Sac1-degron cells treated for 2 h with DMSO, IAA, IAA plus cholesterol, or IAA plus PI4K inhibitors (NC03 and PIK93). n = 3 technical replicates. g Representative confocal images of A549 Sac1-degron cells treated for 2 h with DMSO, IAA, IAA in combination with cholesterol or PI4K inhibitors. Stained for ATP6V 1 A and ATP6V 0 a2. h Violin plot depicting the distribution of the Pearson’s correlation coefficient between ATP6V 1 A and ATP6V 0 a2 from ( g ). Data from one of three independent experiments. One-way ANOVA with Tukey’s multiple comparisons tests. *** p = 0.0008, **** p < 0.0001.

Article Snippet: Plasmids harboring wild-type Sac1 (Sac1 wt ) and a phosphatase-dead mutant Sac1 (C389S, Sac1 mut ) were obtained from Addgene (#108127 and #108128, respectively).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, Western Blot, Concentration Assay

Journal: Nature Communications

Article Title: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

doi: 10.1038/s41467-025-63125-7

Figure Lengend Snippet: a Density plot of gnomAD dataset showing the frequency of genes (y-axis) of having the probability of being loss-of-function intolerant (lof.pLI, x-axis). The orange dotted line indicates the 0.9 threshold commonly used for Mendelian diseases and the red dotted line the location of the SACM1L gene. b Schematic overview of differentiation of trophoblasts from Sac1-degron hESCs. Sac1-degron hESCs are subsequently differentiated into trophoblasts (TB) and syncytiotrophoblasts (STB). IAA treatment can induce Sac1 degradation in both TB and STB. c Representative confocal images of Sac1-degron STB treated for 4 h with IAA and stained for GM130 and TGN46. d Western blot analysis of Sac1-degron STB treated for 4 h with DMSO or IAA and 50 µM EN6 and blotted for TGN46 and B4GALT1. e Bar graph depicting the TGN46 or B4GALT1 degradation efficiency calculated from ( d ). Data from 3 biological replicates. One-way ANOVA with Tukey’s multiple comparisons test. TGN46: n.s. p = 0.7079, * p = 0.0112, p = 0.0278. B4GALT1: ** p = 0.005, **** p < 0.0001. f Violin plot showing the distribution of average PI(4)P staining intensity in the TGN. n > 100 cells. One-way ANOVA with Dunnett’s multiple comparisons test. **** p < 0.0001. g Violin plot showing normalized filipin intensity in the TGN area (excluding lysosomal signal). Data from 3 independent experiments. Two-tailed Mann-Whitney U test. **** p < 0.0001. h Representative confocal images of Sac1-degron STBs treated for 2 h with DMSO or IAA, stained for ATP6V 0 a2 (green) and ATP6V 1 A (magenta). i Western blot analysis of cell lysate and secreted medium of Sac1-degron STB treated with DMSO or IAA for 15 h, with or without Neuraminidase A (Neu A) digestion and blotted for hCGβ. n = 3 independent experiments. j Western blot analysis of secreted medium of Sac1-degron STBs treated with DMSO, IAA or IAA for 15 h, with or without Neuraminidase A digestion. Blotted for hCGβ. n = 3 independent experiments.

Article Snippet: Plasmids harboring wild-type Sac1 (Sac1 wt ) and a phosphatase-dead mutant Sac1 (C389S, Sac1 mut ) were obtained from Addgene (#108127 and #108128, respectively).

Techniques: Staining, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

doi: 10.1038/s41467-025-63125-7

Figure Lengend Snippet: Sac1 fuelled PI(4)P/cholesterol exchange maintains the composition of the Golgi membrane, allowing V-ATPase assembly for TGN acidification, proper terminal glycosylation and cargo secretion. Rapidly after Sac1 degradation, PI(4)P is accumulated in the TGN while cholesterol is depleted. This changes the lipid microenvironment of the V 0 region, resulting in a conformational change in the V 0 a2 subunit which prevents the assembly of the Golgi V-ATPase complex. This results in the deacidification of the TGN, Golgi fragmentation and defects in cargo processing and secretion.

Article Snippet: Plasmids harboring wild-type Sac1 (Sac1 wt ) and a phosphatase-dead mutant Sac1 (C389S, Sac1 mut ) were obtained from Addgene (#108127 and #108128, respectively).

Techniques: Membrane, Glycoproteomics

Journal: Journal of Fungi

Article Title: Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora

doi: 10.3390/jof7090682

Figure Lengend Snippet: Volcano plot of putative interacting proteins from POM33-TagRFP-T pulldowns. For the pulldowns, in the first experiment three biological replicates of Δpom33::pom33-TagRFP-T ect and wt::TagRFP-T ect as control and for the second experiment three biological plus one technical replicate each of the sample and control strain were grown in liquid BMM media over 3 days at 27 °C under continuous light. Protein extracts of the strains were subjected to TagRFP-T-trap pulldown, trypsin digested and resulting peptides were analyzed by LC/MS. The result of two independent experiments is shown. In the volcano plot, the difference of LFQ intensities of POM33-TagRFPT in comparison to free TagRFP-T is plotted on the x-axis versus -Log p -values on the y-axis using a false discovery rate of 0.01 and an S0 of 0.1. The cutoff curve indicates proteins that were significantly enriched with POM33-TagRFP-T. Missing values were replaced four times with imputed values to get reliable interaction candidates. Proteins that were significant in all four repetitions are visible in the upper right part and are marked by dark blue squares (see also ). They include NCA1 (light blue diamond), RTN2 (pink diamond), SEY1 (red diamond), TCB3 (purple diamond), IST2 (yellow diamond) and SAC1 (magenta diamond) as putative POM33 (neon green circle) interaction partners.

Article Snippet: Besides, proteins involved in lipid metabolism, like a cyclopropane-fatty-acyl-phospholipid synthase (SMAC_02133), or the putative phosphatidyl inositol 4-phosphatase SAC1 (SMAC_01583) were determined.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Comparison