Abstract: The mitochondrial-associated endoplasmic reticulum membrane (MAM) is an essential component of eukaryotic cells. It is involved in many critical cellular functions, such as bioenergetics, mitophagy, apoptosis, and calcium signaling, regulating cellular physiological activities. The MAM proteins of mammalian cells have been extensively studied. However, as an important model organism of eukaryotic single cell, only a few MAM proteins were found in yeast, named endoplasmic reticulum and mitochondria encounter structures (ERMES). There is currently a lack of systematic analysis of MAM protein in yeast. Here, the MAM proteins in Saccharomyces cerevisiae that grow under different nutritional conditions were comprehensively identified by using Percoll gradient differential centrifugation and label-free quantification mass spectrometry technique. Finally, 1 486 MAM component proteins are obtained, including well-known MAM proteins and more new components. The functional enrichment analysis of these proteins showed that they are mainly enriched in protein synthesis and transport, glucose metabolism, and lipid metabolism. They are located primarily in the endoplasmic reticulum, mitochondria, Golgi apparatus, and other organelles. Their molecular functions are mainly related to transferase, oxidoreductase activity, proton-transporting ATPase activity, and protein and lipid binding. KEGG metabolic pathway analysis indicated that these proteins participate in sugar biosynthesis, fatty acid metabolism, steroid biosynthesis, endoplasmic reticulum protein processing, amino acid biosynthesis, and other processes. Under different nutritional conditions, the proteins related to mitochondrial division and fusion were identified, such as CAF4, FIS1, DNM1, FZO1 and UGO1. Except for regulatory effects on the structure of mitochondrial, the MAM proteins also have other functions, such as vesicular transport (SSO2, GOS1, VAM6), ion transport (RSN1, YVC1, ATP18), sterol synthesis (ERG1, ERG2, ERG3) and autophagy (ATG39, ATG15, ATG22). Through differential expression analysis, 176 differentially expressed proteins (DEPs) are found under starvation conditions, including 39 up-regulated and 137 down-regulated proteins; 158 DEPs are found under overnutrition conditions, including 155 up-regulated and 3 down-regulated proteins. By analyzing the protein-protein interaction network of DEPs, it was found that they are mainly involved in the biosynthesis of sugars, proton-transporting ATPase activity, GPI-anchored protein biosynthesis, steroid biosynthesis, protein processing in the endoplasmic reticulum, and mitochondrial redox reactions. The pathways and critical proteins involved in sterol synthesis and protein processing were determined, such as MCD4 and CNE1. This research provides significant data resources for further exploring the biological functions of MAM proteins and helps to understand their roles during the process of cellular stress.