Limits...
Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast.

Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D - Mol. Syst. Biol. (2007)

Bottom Line: Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase.Our enzymatic approach was complemented by a parallel protein microarray protein interaction study.Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

Show MeSH
Sequence logos for substrates of Rsp5. PPxY motifs, together with six residues upstream and downstream of the motif from proteins identified as substrates of Rsp5, were aligned and used to generate sequence logos (Crooks et al, 2004). In each logo, stacks of letters indicate the relative frequency of certain amino acids at each position in the sequence. The overall height provides a guide to the level of sequence conservation associated at that position.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1911201&req=5

f3: Sequence logos for substrates of Rsp5. PPxY motifs, together with six residues upstream and downstream of the motif from proteins identified as substrates of Rsp5, were aligned and used to generate sequence logos (Crooks et al, 2004). In each logo, stacks of letters indicate the relative frequency of certain amino acids at each position in the sequence. The overall height provides a guide to the level of sequence conservation associated at that position.

Mentions: We examined the amino-acid sequences of Rsp5 substrates to determine whether additional amino-acids residues in the PY motif ((L/P)PxY) may contribute to substrate specificity. A total of 38 PY motifs were present in 29 proteins of the high-confidence subset. Considering the third (x) position in the motif, the most frequent motifs were PPSY (ten), PPAY (five) and PPPY (five). Comparisons with sets of randomly selected proteins containing PY motifs showed that Ser and Ala (but not Pro) were both significantly overrepresented at the third position within our experimentally determined Rsp5 substrates (P<0.001; randomized exact test) (Figure 3).


Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast.

Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D - Mol. Syst. Biol. (2007)

Sequence logos for substrates of Rsp5. PPxY motifs, together with six residues upstream and downstream of the motif from proteins identified as substrates of Rsp5, were aligned and used to generate sequence logos (Crooks et al, 2004). In each logo, stacks of letters indicate the relative frequency of certain amino acids at each position in the sequence. The overall height provides a guide to the level of sequence conservation associated at that position.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1911201&req=5

f3: Sequence logos for substrates of Rsp5. PPxY motifs, together with six residues upstream and downstream of the motif from proteins identified as substrates of Rsp5, were aligned and used to generate sequence logos (Crooks et al, 2004). In each logo, stacks of letters indicate the relative frequency of certain amino acids at each position in the sequence. The overall height provides a guide to the level of sequence conservation associated at that position.
Mentions: We examined the amino-acid sequences of Rsp5 substrates to determine whether additional amino-acids residues in the PY motif ((L/P)PxY) may contribute to substrate specificity. A total of 38 PY motifs were present in 29 proteins of the high-confidence subset. Considering the third (x) position in the motif, the most frequent motifs were PPSY (ten), PPAY (five) and PPPY (five). Comparisons with sets of randomly selected proteins containing PY motifs showed that Ser and Ala (but not Pro) were both significantly overrepresented at the third position within our experimentally determined Rsp5 substrates (P<0.001; randomized exact test) (Figure 3).

Bottom Line: Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase.Our enzymatic approach was complemented by a parallel protein microarray protein interaction study.Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

Show MeSH