Kre6 Protein Essential for Yeast Cell Wall β-1,6-Glucan Synthesis Accumulates at Sites of Polarized Growth

Abstract

Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for ␤-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesiclelike compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in ␤-1,6-glucan synthesis. This is the first evidence of a ␤-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.

Genome-wide analysis of cell wall-related genes in Tuber melanosporum

“…In S. cerevisiae, where the synthesis mechanism was discovered, ten different enzymes were identified as being involved in this process (Shahinian and Bussey 2000). S. cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and it is essential for β-1,6-glucan synthesis, as revealed by the mutant phenotype, but its biochemical function is still unknown (Kurita et al 2011). Kre6 was first reported as a Golgi protein, but has recently been located mostly in the ER and also in a small but significant portion, in the secretory vesicle-like compartment and plasma membrane (Kurita et al 2011).…”

“…S. cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and it is essential for β-1,6-glucan synthesis, as revealed by the mutant phenotype, but its biochemical function is still unknown (Kurita et al 2011). Kre6 was first reported as a Golgi protein, but has recently been located mostly in the ER and also in a small but significant portion, in the secretory vesicle-like compartment and plasma membrane (Kurita et al 2011). Four homologous genes have been found in the T. melanosporum genome: TmelKre6, TmelKre9, TmelROT1 and TmelSKN1.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…As hydrolases or transglycosylases, Kre6 and Skn1 could act on a structure that serves as a precursor or acceptor in elaboration of b1,6-glucan or on a glycoprotein involved in b1,6-glucan synthesis (Lesage and Bussey 2006), but enzyme activity has yet to be demonstrated for these proteins. Much of Kre6 is ERlocalized, where it interacts with Keg1, but the protein is also detectable in the Golgi, in secretory vesicles, and at the plasma membrane at sites of polarized growth Nakamata et al 2007;Kurita et al 2011;File S8). Localization of Skn1 has not been explored in detail.…”

“…Recent studies indicate that much of Kre6 is ER--localized, where it interacts with Keg1, but Kre6 is also detectable in secretory vesicles and at the plasma membrane at sites of polarized growth (Nakamata et al 2007;Kurita et al 2011). In addition to Kre6's lumenal domain, the protein's cytoplasmic tail is important for Kre6's function in β1,6--glucan assembly and its transport to the plasma membrane Kurita et al 2011). A truncated form of Kre6 lacking its 230 N--terminal amino acids failed to be localized to the plasma membrane, and did not correct the β1,6--glucan synthetic defect of kre6Δ, although it appeared stable .…”

Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

“…Some of these cargo proteins require the assistance of cytosolic or membrane-spanning accessory adaptor proteins for their incorporation into COPII vesicles. Several adaptor proteins have been identified to assist the COPII machinery in yeast (3)(4)(5); however, fewer have been characterized in higher eukaryotes. In metazoans, ERGIC-53 mediates the export of blood clotting factors, Cathepsin Z and C and α-1 antitrypsin (6), and SCAP [sterol-regulatory elementary binding protein (SREBP) cleavage activating protein] mediates the regulated transport of SREBP protein from the ER to the Golgi in cells that are sterol-deficient (7).…”