All cellular functions rely on accurate protein biosynthesis. Yet, many variants of transfer RNA (tRNA) genes that induce amino acid misincorporation are found in human genomes. Mistranslation induces pleiotropic effects on proteostasis, ranging from protein misfolding to impaired protein biosynthesis and degradation. We employ Saccharomyces cerevisiae (budding yeast), a genetically and biochemically tractable model that facilitates quantitative analysis of how specific proteostasis pathways interact with mistranslating tRNAs. We tested two mistranslating tRNASer variants, one inducing proline to serine (P > S), the other arginine to serine (R > S) misincorporation. We found that P > S misincorporation impairs cellular fitness and sensitizes cells to protein misfolding to a greater extent than R > S misincorporation. Of note, we also show that, even though both tRNA variants induce misincorporation of serine, they result in the accumulation of misfolded proteins by distinct mechanisms. Specifically, R > S misincorporation reduces that association of Hsp70 with misfolded proteins, while P > S misincorporation impairs the degradation of nascent polypeptides. Our findings reveal that different mistranslating tRNASer variants impair specific branches of proteostasis and thus compromise cellular fitness by distinct mechanisms.