Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

Abstract

Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected approximately 1100 peptides and their approximately 200 protein-substrate origins we obtain evidence for new insights into the proteome-wide protein-selective degradation in yeast cells. This evidence shows that the yeast cytoplasm is the largest pool for the degradation of proteins with both biochemical and geometric specificities, whereas the yeast nucleus seems to be a proteolysis-inert organelle under the condition studied. Yeast V-ATPase subunits appear to be degraded during their disassembly, and yeast mitochondrial proteins functioning as precursors, transport carriers, and gates are preferentially degraded. Ubiquitylation may be unnecessary for the proteasomal degradation of yeast cytoplasmic regulatory and enzyme proteins according to our observations. This study shows that the intracellular peptides are informational targets for directly probing the protein degradation-involved molecular mechanisms and cell biology processes.

Figure 1

LC-MS display of molecular species having molecular masses of 400–10,000 u in a yeast Saccharomyces cerevisiae lysate. Species with >3 MS scans were counted. The mass accuracies for both amino acid sequencing and individual mass peaks are illustrated. The de novo sequencing-based interpretation of high-precision MS/MS and experimental conditions are described in detail in the Methods section.

Figure 2

Peptide ladders showing yeast phosphoglycerate kinase sequences occurring in the cells at the time of cell harvest. The peptides observed had lengths of 12–70 amino acid residues; the curved sequence unnecessarily represents the protein real folding.

Figure 3

Localization of 118 yeast proteins observed with the existence of their intracellular peptides. Proteins observed in each organelle are described in the following sections.

Figure 4

Proteolytic cleavage preferences of yeast Saccharomyces cerevisiae cytoplasmic proteins. 462 peptides observed for the 77 cytoplasm-specific proteins were analyzed. The cleavage sites were defined as follows: NH2- … –P1 ↓ P1′– …COOH, where the arrow represents the proteolytic cleavage site. a) The percentage (or frequency) of amino acids at the two cleavage sites; b) the relative frequency (%) = [(percentage of the amino acid in all detected P1 or P1′ termini – percentage of the amino acid in detected proteins)/percentage of the amino acid in detected proteins] × 100.

Figure 5

Observed degradation of the yeast V-ATPase subunits suggesting that the process happens after disassembly of subunits from subunit D. The V-ATPase structure diagram was redrawn based on ref. (33).

Figure 6

Distribution of the degraded yeast mitochondrial proteins. The translocation diagram was modified based on ref. (35).