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Keratin 1 plays a critical role in golgi localization of core 2 N-acetylglucosaminyltransferase M via interaction with its cytoplasmic tail.

ABSTRACT: Core 2 N-acetylglucosaminyltransferase 2/M (C2GnT-M) synthesizes all three ?6GlcNAc branch structures found in secreted mucins. Loss of C2GnT-M leads to development of colitis and colon cancer. Recently we have shown that C2GnT-M targets the Golgi at the Giantin site and is recycled by binding to non-muscle myosin IIA, a motor protein, via the cytoplasmic tail (CT). But how this enzyme is retained in the Golgi is not known. Proteomics analysis identifies keratin type II cytoskeletal 1 (KRT1) as a protein pulled down with anti-c-Myc antibody or C2GnT-M CT from the lysate of Panc1 cells expressing bC2GnT-M tagged with c-Myc. Yeast two-hybrid analysis shows that the rod domain of KRT1 interacts directly with the WKR(6) motif in the C2GnT-M CT. Knockdown of KRT1 does not affect Golgi morphology but increases the interaction of C2GnT-M with non-muscle myosin IIA and its transportation to the endoplasmic reticulum, ubiquitination, and degradation. During Golgi recovery after brefeldin A treatment, C2GnT-M forms a complex with Giantin before KRT1, demonstrating CT-mediated sequential events of Golgi targeting and retention of C2GnT-M. In HeLa cells transiently expressing C2GnT-M-GFP, knockdown of KRT1 does not affect Golgi morphology but leaves C2GnT-M outside of the Golgi, resulting in the formation of sialyl-T antigen. Interaction of C2GnT-M and KRT1 was also detected in the goblet cells of human colon epithelial tissue and primary culture of colonic epithelial cells. The results indicate that glycosylation and thus the function of glycoconjugates can be regulated by a protein that helps retain a glycosyltransferase in the Golgi.

SUBMITTER: Petrosyan A

PROVIDER: S-EPMC4358263 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Keratin 1 plays a critical role in golgi localization of core 2 N-acetylglucosaminyltransferase M via interaction with its cytoplasmic tail.

Petrosyan Armen A Ali Mohamed F MF Cheng Pi-Wan PW

The Journal of biological chemistry 20150120 10

Core 2 N-acetylglucosaminyltransferase 2/M (C2GnT-M) synthesizes all three β6GlcNAc branch structures found in secreted mucins. Loss of C2GnT-M leads to development of colitis and colon cancer. Recently we have shown that C2GnT-M targets the Golgi at the Giantin site and is recycled by binding to non-muscle myosin IIA, a motor protein, via the cytoplasmic tail (CT). But how this enzyme is retained in the Golgi is not known. Proteomics analysis identifies keratin type II cytoskeletal 1 (KRT1) as ...[more]

PMID: 25605727

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Project description:Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

Dataset Information

Keratin 1 plays a critical role in golgi localization of core 2 N-acetylglucosaminyltransferase M via interaction with its cytoplasmic tail.

Publications

Keratin 1 plays a critical role in golgi localization of core 2 N-acetylglucosaminyltransferase M via interaction with its cytoplasmic tail.

Similar Datasets

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