Protein N-myristoylation plays a critical role in the endoplasmic reticulum morphological change induced by overexpression of protein Lunapark, an integral membrane protein of the endoplasmic reticulum.

Koko Moriya,Takashi Suzuki,Toshihiko Utsumi,Yoshimi Noriyasu,Emi Takamitsu,Kei Nagatoshi,Tsuyoshi Okamura,F Gisou Van Der Goot

doi:10.1371/journal.pone.0078235

Koko Moriya, Takashi Suzuki + Show 6 more

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https://doi.org/10.1371/journal.pone.0078235

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Journal: PloS one	Publication Date: Nov 4, 2013
Citations: 93	License type: CC BY 4.0

Affiliation: Yamaguchi University, Shimadzu (Japan)

Abstract

N-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human N-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an N-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein N-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein N-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.

Highlights

Protein N-myristoylation is the attachment of myristic acid, a 14-carbon saturated fatty acid, to the N-terminal Gly of proteins [1,2,3,4,5]
A recent report revealed that Lnp1p, the yeast ortholog of protein Lunapark, is a membrane protein of the endoplasmic reticulum (ER) and is involved in network formation of the ER [28]
When these cDNAs were transfected to COS-1 cells and metabolic labeling with [3H]myristic acid was performed, only two proteins (KIAA1609 and protein Lunapark) were expressed efficiently, as determined by the Western blotting analysis of total cell lysates of the transfected cells (Fig. 1C, left panel)

Summary

Introduction

Protein N-myristoylation is the attachment of myristic acid, a 14-carbon saturated fatty acid, to the N-terminal Gly of proteins [1,2,3,4,5]. In addition to the cotranslational protein N-myristoylation, it is established that posttranslational N-myristoylation can occur on many caspase-cleavage products in apoptotic cells [6,7,8] Both cotranslational and posttranslational N-myristoylation are catalyzed by N-myristoyltransferase (NMT), a member of the GCN5related N-acetyltransferase superfamily of proteins [9]. Many Nmyristoylated proteins play key roles in regulating cellular structure and function. They include proteins involved in a wide variety of cellular signal transduction pathways, such as protein kinases, phosphatases, guanine nucleotide binding proteins, Ca2+

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