Large-scale comparative genomics in harness with proteomics has substantiated fundamental features of eukaryote cellular evolution. The evolutionary trajectory of modern eukaryotes is distinct from that of prokaryotes. Data from many sources give no direct evidence that eukaryotes evolved by genome fusion between archaea and bacteria. Comparative genomics shows that, under certain ecological settings, sequence loss and cellular simplification are common modes of evolution. Subcellular architecture of eukaryote cells is in part a physical-chemical consequence of molecular crowding; subcellular compartmentation with specialized proteomes is required for the efficient functioning of proteins.