Peroxisomes are common eukaryotic cell organelles. So far, the inventory of the yeast peroxisomal proteome is still incomplete. To identify novel peroxisomal proteins, we compared yeast wild-type (WT) and PEX3 knockout cells, which lack functional peroxisomes by whole cell proteomics and transcriptomics. Cells were grown on acetate, a carbon source that involves peroxisomal enzymes of the glyoxylate cycle. Transcriptome analysis showed that, with the exception of transcripts of β-oxidation enzymes, the transcriptome of peroxisomal proteins was largely unchanged. In contrast, whole cell proteomics analysis revealed that peroxisome deficiency results in a reduction of the levels of peroxisomal membrane proteins. A set of non-peroxisomal membrane proteins showed a similar behavior. We therefore analyzed one of these proteins, the mitochondrial pyruvate carrier protein Mpc3, and show that Mpc3 has a dual localization on peroxisomes and mitochondria. In conclusion we present a novel approach to uncover new examples of dually localized membrane proteins.