Anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loadingAnti-Gap1 antibody. Bottom panels: Western

Anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading
Anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading handle. Luminescent arbitrary units (LAU) 10-6 are shown as ratio involving the Gap1 band and Pma1 band for every single time point to Cytochrome c/CYCS Protein MedChemExpress assess relative disappearance of the Gap1 band, constant with endocytosis. The ratios in between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative raise from the former with respect to the latter following addition of every nitrogen supply. A Western blot from cells expressing the non-ubiquitinatable Gap1K9R,K16R subjected to identical treatment is also shown as control to confirm that upper bands observed above the Gap1 band within the wild-type blots are ubiquitinated types with the transceptor.2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 93, 213222 G. Van LacI Protein supplier Zeebroeck, M. Rubio-Texeira, J. Schothorst and J. M. Thevelein2014 The Authors. Molecular Microbiology published by John Wiley Sons Ltd., Molecular Microbiology, 93, 213Analogues uncouple transceptor functionsFig. 5. The non-transported and non-signalling competitive inhibitor of Gap1-mediated transport, L-Asp–L-Phe, can’t trigger endocytosis but triggers ubiquitination inside the wild-type strain. A. Progressive intracellular accumulation of radioactively labelled L-Asp–L-Phe after addition of 5 mM of this compound to nitrogen-starved cells. Strains: wild-type (black bars), gap1 (white bars) and opt1 dal5 ptr2 (grey bars). Error bars represent s.d. amongst biological repeats. B. Growth of 110 serial dilution spottings of nitrogen pre-starved cells of the strains wild-type, gap1, opt1 dal5 ptr2 and opt1 dal5 ptr2 gap1 on plates of nitrogen starvation medium (NSM) without the need of or supplemented with 1 mM of L-citrulline, or L-Asp–L-Phe. The exact same cells spotted in comprehensive supplemented medium (CSM) are shown as positive development handle. Growth from the similar cells in NSM 1 mM with the dipeptide Leu-Met-NH2 or the tripeptide L-Arg-Gly-Gly is shown as control of peptide use as nitrogen supply as a consequence of peptide carrier uptake. C. Localization of wild-type Gap1-GFP expressed inside the strains gap1 and opt1 dal5 ptr2 gap1 is shown prior to and 60, 120 and 180 min just after addition of 5 mM L-Asp–L-Phe. The identical cells exposed to 2.five mM L-aspartate plus 2.5 mM L-phenylalanine is shown as handle that the dipeptide constituent amino acids are able to induce endocytosis. D. Analysis of Gap1 ubiquitination status in nitrogen-starved cells expressing endogenous GAP1 (from the wild-type or the triple deletion mutant opt1 dal5 ptr2) and induced with 10 M CuSO4 for 30 min before addition of nitrogen supply, for expression of myc-ubiquitin from the PCUP1-myc-Ubi URA3 plasmid, pMRT7. P13 fractions had been collected at unique time points (0, 30, 60, 120 and 180 min) following addition of five mM L-Asp–L-Phe to nitrogen-starved cells. Upper panels: Western blot with anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading control. Luminescent arbitrary units (LAU) 10-6 are shown as ratio amongst the Gap1 band and Pma1 band for each time point to assess the relative disappearance from the Gap1 band, consistent with endocytosis. The ratios involving di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative raise from the former with respect to the latter soon after addition of every nitrogen source.2002; Merhi and Andr 2012). Transport was fully abolished by deletion on the 3 major pe.