Control of the Yeast Cell Cycle with a Photocleavable α‐Factor Analogue

Abstract

The budding yeast Saccharomyces cerevisiae is widely used as a model organism for studying many biological processes and systems, particularly for understanding the cell cycle. The yeast peptide pheromone α-factor (WHWLQLKPGQPMY) activates the mating pathway in MATa cells, arresting the cell cycle in the G1 phase. This arrest is used as a tool for synchronizing yeast cultures and for investigating signaling events involved in the pathway and other processes related to morphogenesis and transcription. Herein we report the synthesis and activity of an α-factor analogue designed to control the on/off state of the mating signal. Incorporating a photocleavable residue at a flexible site in the peptide allowed UV degradation in situ to mimic the natural protease degradation of this signaling peptide. The control over the α-factor signal achieved serves as proof of concept for use of this analogue as a tool. It also and allows access to previously impractical experiments that will yield highly time-resolved information about the yeast cell cycle and mating pathway.

Photocleavable peptide–oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

“…44, 56–66 While chemically cleavable bifunctional cross-linkers are readily available, the previously described photocleavable cross-linkers have not been commercialized and require significant synthetic efforts. As a starting point, we chose sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate), a commercially available, water-soluble, heterobifunctional cross-linker with amine and sulfhydryl reactivity.…”

“…Approaches based on exploiting photocleavable crosslinkers have proven to offer a rapid and efficient route for syntheses of many photocleavable bioconjugates for applications in diagnostics and therapeutics. 44,[56][57][58][59][60][61][62][63][64][65][66] While chemically cleavable bifunctional cross-linkers are readily available, the previously described photocleavable cross-linkers have not been commercialized and require significant synthetic efforts. As a starting point, we chose sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), a commercially available, water-soluble, heterobifunctional cross-linker with amine and sulfhydryl reactivity.…”

“…[40][41][42] Applying this principle to homogeneous assays, Kubota et al 32 developed a single-reaction strategy to measure the differential phosphorylation in 90 peptide substrates using electrospray ionization mass spectrometry. We 24,25,[43][44][45] and others [46][47][48] have exploited matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a tool for label-free detection of peptide phosphorylation in heterogeneous assays with substrates tethered to MALDI targets, self-assembled monolayers, microarrays, microparticles and other geometries. MALDI-TOF MS-based phosphorylation detection is comparatively simple, does not require elaborate chromatography-based separation steps, is compatible with HTS and has the potential for unlimited multiplexing.…”

Development of Chemical Biology Tools Focusing on Peptide/Amide Bond Cleavage Reaction

“…3,4) Several caged peptides and proteins, which undergo UV-responsive main chain cleavage to alter their bioactivities, were developed to clarify mechanisms of action of original uncaged peptides/ proteins. [5][6][7][8] These caged peptides/proteins contain a UVresponsive artificial amino acid such as 2-nitrophenylglycine; therefore laborious de novo design and synthesis of a new amino acid will be required if the peptides/proteins should re-spondtoastimulusotherthanUV.Inthiscontext,westarted to invent a stimulus-responsive peptide-bond-cleaving residue (Spr), which can respond to various stimuli by just replacing its protecting group 2) ( Fig. 1A).…”

Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

“…Photoactivatable enzymes and enzyme substrates are well-established tools for the investigation of biological function and catalytic mechanism (15)(16)(17)(18)(19)(20). Using a photocleavable yeast peptide pheromone (a-factor, WHWLQLKPGQPMY), Parker et al (21) recently demonstrated that the arrest of MATa cells at the G1 stage of division is possible with brief exposure to light. Such control allows for synchronizing yeast cell cultures and investigating signaling processes related to morphogenesis and transcription.…”