Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include Regulators of the Actin Cytoskeleton

Abstract

Transmembrane mucin-type glycoproteins can regulate signal transduction pathways. In yeast, signaling mucins regulate mitogen-activated protein kinase (MAPK) pathways that induce cell differentiation to filamentous growth (fMAPK pathway) and the response to osmotic stress (HOG pathway). To explore regulatory aspects of signaling mucin function, protein microarrays were used to identify proteins that interact with the cytoplasmic domain of the mucin-like glycoprotein, Msb2p. Eighteen proteins were identified that comprised functional categories of metabolism, actin filament capping and depolymerization, aerobic and anaerobic growth, chromatin organization and bud growth, sporulation, ribosome biogenesis, protein modification by iron-sulfur clusters, RNA catabolism, and DNA replication and DNA repair. A subunit of actin capping protein, Cap2p, interacted with the cytoplasmic domain of Msb2p. Cells lacking Cap2p showed altered localization of Msb2p and increased shedding of Msb2p's N-terminal glycosylated domain. Consistent with its role in regulating the actin cytoskeleton, Cap2p, and another Msb2p-interacting protein, Aip1p, were required for the enhanced cell polarization during filamentous growth. Our study identifies proteins that connect a signalling mucin to diverse cellular processes and may provide insight into new aspects of mucin function. Msb2p Interacting Proteins Prabhakar et al. 3 Msb2p Interacting Proteins Prabhakar et al. 4 many fungal species 24, 25 . Cells undergoing filamentous growth grow as branched filaments of elongated and connected cells 26-29 . One of the pathways that regulates filamentous growth is a Cdc42p-dependent Mitogen Activated Protein Kinase (MAPK) pathway commonly referred to as the fMAPK pathway 30, 31 . Msb2p functions at the plasma membrane to regulate the fMAPK pathway 4, 5 . Msb2p is a single-pass transmembrane protein with a highly glycosylated N-terminal domain (1185 amino acids) connected to a cytoplasmic C-terminal signaling domain (97 amino acids) by a transmembrane domain. The cytoplasmic domain of Msb2p binds directly to Cdc42p 20 . Cdc42p associates with the p21-activated kinase (PAK) Ste20p 32, 33 to regulate the fMAPK cascade [Ste11p, Ste7p, and Kss1p 34 ] that culminates in the phosphorylation/activation of transcription factors [Ste12p and Tec1p 35 ], which induce the expression of filamentation target genes 36, 37 . The glycosylation of Msb2p is related to its signaling function 38 . Under nutrient-limiting conditions, Msb2p is underglycosylated, which results in its proteolytic processing through a quality-control pathway called the Unfolded Protein Response (UPR) in the lumen of ER. The UPR upregulates the expression of an aspartyl protease, Yps1p that processes Msb2p in its extracellular domain 39, 40 . Proteolytic processing of Msb2p is required for activation of of the fMAPK pathway 40 . Another signaling mucin in yeast is Hrk1p, which regulates the Ste11p branch of the HOG pathway 41 . The HOG pathway has two branches that converge on the MAPKK Pbs2p. Hrk...