Previously, we have shown that single-gene overexpression of five yeast genes (CCW12, CWP2, ERO1, RPPO, and SEDI) promoted increased secretion levels of several single-chain antibody fragments and single-chain T-cell receptors from Saccharomyces cerevisiae (Wentz, A. E.; Shusta, E. V. Appl. Environ. Microbiol. 2007, 73, (4), 1189-1198). In this study, several proteins possessing different protein folds were secreted from yeast overexpressing each of the five genes to determine the generality of the secretion enhancers. Only one gene encoding a ribosomal subunit (RPPO) enhanced secretion levels for multiple proteins: a single-chain antibody (the 4-4-20 anti-fluorescein scFv) and green fluorescent protein (GFP). Protein induction time-course experiments revealed increased secretion with RPPO overexpression for 4-4-20 as early as 40 h post-induction. Effects on GFP secretion levels were not evident until late induction times where overexpression of RPPO limited post-secretion protein loss, but absolute yields did not exceed those observed at earlier induction times. The effects of RPPO overexpression on secreted protein yields did not appear to directly involve ribosome function, but instead RPPO overexpression indirectly regulated acidification of the yeast medium by preventing upregulation of the yeast plasma membrane H+-ATPase gene, PMA1. Combining RPPO overexpression with nutrient supplementation stimulated additional protein secretion for the 4-4-20 scFv with higher per cell secretion that corresponded to 6-fold increases in volumetric yield.