A modified KESTREL search reveals a basophilic substrate consensus for the Saccharomyces cerevisiae Npr1 protein kinase.

The Saccharomyces cerevisiae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the Tor (target of rapamycin) signaling pathway. Inhibition of the Tor proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. As an alternative to peptide arrays, the substrate requirement of Npr1 was probed with a peptide library that was generated by cleaving yeast cell extracts with CNBr, and after reverse-phase chromatography, the individual fractions were phosphorylated in vitro with recombinant Npr1. In this way, the ribosomal protein Rpl24a was found to be an excellent in vitro substrate for Npr1. Synthetic peptides tailored around the phosphorylation site of Rpl24a show that Npr1 is a Ser/Thr protein kinase with an absolute requirement for a basic residue at the P-3 position and a strong preference for basic P + 1 residues, whereas proline at P + 1 is strongly disfavored. The results obtained with synthetic peptides suggest a (K/R)-X-X-S-(K/R) consensus sequence for Npr1. The availability of a consensus sequence allows a targeted search for physiologically relevant Npr1 substrates involved in the regulation of yeast amino acid permeases.

Identification of the rapamycin-sensitive phosphorylation sites within the Ser/Thr-rich domain of the yeast Npr1 protein kinase.

The Saccharomyces cerevisae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a fungus-specific family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the TOR (target of rapamycin) signalling pathway. Inhibition of the TOR proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. To identify the rapamycin-sensitive phosphorylation sites in yeast Npr1, glutathione-S-transferase (GST)-tagged Npr1 was isolated from untreated or rapamycin-treated cells, and analyzed by mass spectrometry. Here, we report for the first time 22 phosphorylation sites that are clustered in two regions of the N-terminal serine-rich domain. All phosphorylation sites, except two, were found to be rapamycin-sensitive. Some phosphorylation sites are contained in motifs that closely resemble those in mammalian S6K (serines followed by a tyrosine or a phenylalanine) and 4E-BP1 (serines followed by a proline). Other sites, such as serines followed by Ala, Asn, Gln, His, Ile, Leu, or Val, appear to define new motifs. Thus, TOR controls an unusually broad array of phosphorylation sites in Npr1. In addition to phosphorylation by upstream kinases, Npr1 undergoes autophosphorylation that was mapped to three distinct serines in the N-terminal domain of which Ser257 appears to be the main autophosphorylation site. Site-directed mutagenesis confirmed the mass spectral assignments of the autophosphorylation sites and shows that Ser257 is specifically involved in forming an in vitro substrate-binding site.

Probing the environment of signal-anchor sequences during topogenesis in the endoplasmic reticulum.

Signal sequences for insertion of protein into the mammalian endoplasmic reticulum orient themselves in the translocon on the basis of their flanking charges. It has recently been shown that hydrophobic N-terminal signals initially insert head-on before they invert their orientation to translocate the C-terminus. The rate of inversion is reduced with the increasing hydrophobicity of the signal due to an increased affinity for the initial bound state at the translocon. To probe the environment of the signal while its orientation is determined, different hydrophobic residues were inserted at various positions throughout a uniform oligoleucine signal sequence and the constructs were expressed in transfected COS-7 cells. The resulting topologies revealed a strikingly symmetric position dependence specifically for bulky aromatic amino acids, reflecting the structure of a lipid bilayer. Maximal N-translocation was observed when the guest residues were placed at the N- or C-terminus of the hydrophobic sequence or in the very center, corresponding to the positions of highest expected affinity of the signal sequence as a membrane-spanning helix for the bilayer. The results support the model that during topogenesis in vivo the signal sequence is exposed to the lipid membrane.