Systems-wide Proteomic Analysis in Mammalian Cells Reveals Conserved, Functional Protein Turnover

Abstract

The turnover of each protein in the mammalian proteome is a functionally important characteristic. Here, we employed high-resolution mass spectrometry to quantify protein dynamics in nondividing mammalian cells. The ratio of externally supplied versus endogenous amino acids to de novo protein synthesis was about 17:1. Using subsaturating SILAC labeling, we obtained accurate turnover rates of 4106 proteins in HeLa and 3528 proteins in C2C12 cells. Comparison of these human and mouse cell lines revealed a highly significant turnover correlation of protein orthologs and thus high species conservation. Functionally, we observed statistically significant trends for the turnover of phosphoproteins and gene ontology categories that showed extensive covariation between mouse and human. Likewise, the members of some protein complexes, such as the proteasome, have highly similar turnover rates. The high species conservation and the low complex variances thus imply great regulatory fine-tuning of protein turnover.

The nature and extent of contributions by defective ribosome products to the HLA peptidome

“…Partially or fully unfolded proteins (4,42,43), products of premature termination of translation (12)(13)(14), downstream initiation (10), products of the pioneer round of translation (15), and out-of-frame translated segments of proteins (18). Another possible source of rapidly degraded proteins are full-sized, surplus subunits of large protein complexes, which are degraded rapidly after being produced in excess of the need for assembly (20,22,42). Our data support this last option as a likely source of the rapidly degraded proteins, which supply a significant portion of the MHC peptidome.…”

“…This conclusion is based on the large contribution of subunits of large complexes, such as the ribosome and TCP-1 subunits, to the high DRiPs factor HLA peptidome. The excess production of such subunits can be caused by limiting production of one or more of the core subunits of the protein complexes (22,44) or in the case of the ribosomes, by limited availability of some ribosomal subunits (45) or rRNA ( Fig. 2 and Table 2).…”

“…MHC peptides derived from newly synthesized proteins were also suggested to be produced by degradation of normal, fully functional polypeptides, which are the excess subunits of large protein complexes (19,20). In those cases, where all of the subunits are not simultaneously ready for assembly, the excess subunits should be degraded to avoid interference with the wellbeing of the cells (21,22).…”

“…The rates of synthesis of cellular proteins can be determined by the use of large-scale dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) experiments (30) or by its newer version-pulsed SILAC (22), both of which are modifications of the classical (static) SILAC approach (31). SILAC is mostly used for mass spectrometry determination of the relative amounts of proteins by metabolic incorporation of stable isotope labeled amino acids.…”

The nuclear pore complex – structure and function at a glance

“…Whereas most mammalian proteins have an average half-life of a few days (Cambridge et al, 2011;Price et al, 2010), recent whole animal pulse-chase experiments demonstrated that, in postmitotic tissues, the structural scaffold of the NPC, or at least components thereof, persists over the entire lifetime of a cell (Toyama et al, 2013). In dividing cells, NPCs undergo a cycle of assembly and disassembly that is in concert with the cell cycle.…”