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Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.

Van Zeebroeck G, Rubio-Texeira M, Schothorst J, Thevelein JM - Mol. Microbiol. (2014)

Bottom Line: L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling.These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism.Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Leuven-Heverlee, Flanders, B-3001, Belgium.

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Behaviour of nearly transport-inactive Gap1Y395C in the presence of non-signalling amino acids l-histidine and l-lysine.A. Transport of 5 mM l-citrulline, l-histidine or l-lysine in wild-type (black bars) or gap1Y395C (white bars) strains. Error bars represent s.d. between biological repeats.B. Gap1Y395C-GFP localization is shown 0, 60, 120 and 180 min after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells.C. Analysis of Gap1 ubiquitination status in nitrogen-starved gap1Δ cells expressing Gap1Y395C (from YCpGap1Y395C, URA3 plasmid) and induced with 10 μM CuSO4 for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the PCUP1-myc-Ubi HIS3 plasmid, pMRT39. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-citrulline, l-histidine or l-lysine 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 between the Gap1 band and Pma1 band for each time point to assess the relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source.
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fig06: Behaviour of nearly transport-inactive Gap1Y395C in the presence of non-signalling amino acids l-histidine and l-lysine.A. Transport of 5 mM l-citrulline, l-histidine or l-lysine in wild-type (black bars) or gap1Y395C (white bars) strains. Error bars represent s.d. between biological repeats.B. Gap1Y395C-GFP localization is shown 0, 60, 120 and 180 min after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells.C. Analysis of Gap1 ubiquitination status in nitrogen-starved gap1Δ cells expressing Gap1Y395C (from YCpGap1Y395C, URA3 plasmid) and induced with 10 μM CuSO4 for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the PCUP1-myc-Ubi HIS3 plasmid, pMRT39. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-citrulline, l-histidine or l-lysine 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 between the Gap1 band and Pma1 band for each time point to assess the relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source.

Mentions: We previously showed that the Gap1Y395C protein, mutated in a residue located in TMDVIII has strongly reduced transport and signalling with regular amino acids (Van Zeebroeck et al., 2009). Transport of l-histidine and l-lysine was also strongly reduced in this mutant (Fig. 6A). When l-citrulline, l-histidine and l-lysine were added to nitrogen-starved cells we did not observe substantial disappearance of Gap1Y395C-GFP from the plasma membrane (Fig. 6B). A similar lack of enhanced internalization was also observed when Gap1Y395C-GFP was exposed to l-asparagine or to the non-metabolizable analogues β-alanine or d-histidine (Fig. S9). Even though the three non-signalling l-amino acids were unable to trigger endocytosis of this mutant form of Gap1, they still elicited oligo-ubiquitination of the mutated transceptor, as observed by detection of newly appearing di- and tri-ubiquitinated Gap1 (Fig. 6C). This further indicates that oligo-ubiquitination of Gap1 is not sufficient to elicit normal rates of endocytosis and that regular rates of transport are not required to trigger oligo-ubiquitination.


Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.

Van Zeebroeck G, Rubio-Texeira M, Schothorst J, Thevelein JM - Mol. Microbiol. (2014)

Behaviour of nearly transport-inactive Gap1Y395C in the presence of non-signalling amino acids l-histidine and l-lysine.A. Transport of 5 mM l-citrulline, l-histidine or l-lysine in wild-type (black bars) or gap1Y395C (white bars) strains. Error bars represent s.d. between biological repeats.B. Gap1Y395C-GFP localization is shown 0, 60, 120 and 180 min after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells.C. Analysis of Gap1 ubiquitination status in nitrogen-starved gap1Δ cells expressing Gap1Y395C (from YCpGap1Y395C, URA3 plasmid) and induced with 10 μM CuSO4 for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the PCUP1-myc-Ubi HIS3 plasmid, pMRT39. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-citrulline, l-histidine or l-lysine 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 between the Gap1 band and Pma1 band for each time point to assess the relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4285233&req=5

fig06: Behaviour of nearly transport-inactive Gap1Y395C in the presence of non-signalling amino acids l-histidine and l-lysine.A. Transport of 5 mM l-citrulline, l-histidine or l-lysine in wild-type (black bars) or gap1Y395C (white bars) strains. Error bars represent s.d. between biological repeats.B. Gap1Y395C-GFP localization is shown 0, 60, 120 and 180 min after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells.C. Analysis of Gap1 ubiquitination status in nitrogen-starved gap1Δ cells expressing Gap1Y395C (from YCpGap1Y395C, URA3 plasmid) and induced with 10 μM CuSO4 for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the PCUP1-myc-Ubi HIS3 plasmid, pMRT39. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-citrulline, l-histidine or l-lysine 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 between the Gap1 band and Pma1 band for each time point to assess the relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source.
Mentions: We previously showed that the Gap1Y395C protein, mutated in a residue located in TMDVIII has strongly reduced transport and signalling with regular amino acids (Van Zeebroeck et al., 2009). Transport of l-histidine and l-lysine was also strongly reduced in this mutant (Fig. 6A). When l-citrulline, l-histidine and l-lysine were added to nitrogen-starved cells we did not observe substantial disappearance of Gap1Y395C-GFP from the plasma membrane (Fig. 6B). A similar lack of enhanced internalization was also observed when Gap1Y395C-GFP was exposed to l-asparagine or to the non-metabolizable analogues β-alanine or d-histidine (Fig. S9). Even though the three non-signalling l-amino acids were unable to trigger endocytosis of this mutant form of Gap1, they still elicited oligo-ubiquitination of the mutated transceptor, as observed by detection of newly appearing di- and tri-ubiquitinated Gap1 (Fig. 6C). This further indicates that oligo-ubiquitination of Gap1 is not sufficient to elicit normal rates of endocytosis and that regular rates of transport are not required to trigger oligo-ubiquitination.

Bottom Line: L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling.These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism.Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Leuven-Heverlee, Flanders, B-3001, Belgium.

Show MeSH