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

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.

Arabidopsis Lunapark proteins are involved in ER cisternae formation

“…The mammalian Lnp (mLnp1) protein features an N‐terminal myristoylation site, and ER network changes induced by overexpression of LNP proteins were significantly inhibited by the mutation of protein N ‐myristoylation which rendered this motif nonfunctional (Moriya et al ., 2013). Yeast Lnp1p protein, by contrast, does not feature this motif (Moriya et al ., 2013).…”

“…Yeast Lnp1p protein, by contrast, does not feature this motif (Moriya et al ., 2013). Arabidopsis LNP proteins similarly do not possess an N‐terminal glycine and therefore are predicted to lack a myristoylation site as shown for mLnp1 (Moriya et al ., 2013). …”

“…Mammalian Lnp1 is dependent on N ‐myristoylation for its localization to ER junctions and morphogenic activity. The absence of this motif in yeast Lnp1p could indicate that LNP proteins in higher organisms have evolved an additional degree of functionality and/or regulation via post‐translational lipid modifications (Moriya et al ., 2013; Wang et al ., 2016; Turnbull & Hemsley, 2017). …”

Developmental- and stress-mediated expression analysis of cinnamoyl-CoA reductase 1 (CCR1) fromHibiscus cannabinus

“…Moreover, co-translational modification of protein is required to carry out some specific functions such as protein trafficking, aggregation, and affinity for membrane attachment (Adamson et al 1992;Löfke et al 2013). These co-translational modifications may be either palmitoylation or myristoylation (Linder & Deschenes 2007;Wright et al 2010;Moriya et al 2013). Sequence analysis revealed the presence of N-terminal palmitoylation sites in HcCCR1 (SSNGMTVCVTGAGGF) and HcCCR2 (CSNGTTVCVTGAGGF) protein.…”

Abscisic Acid Transport in Human Erythrocytes

“…The LANCL2 protein is associated with the plasma membrane through N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site (21). However, protein lipidation, a typical feature of peripheral membrane proteins, has been recently observed in integral membrane proteins as well (22), and previous immunofluorescence studies performed on LANCL2-overexpressing cells were inconclusive regarding the transmembrane or peripheral position of LANCL2 (18). Here, we investigated LANCL2 localization in human erythrocytes, and found that LANCL2 is a peripheral protein attached to the intracellular side of the RBC membrane; thus, for ABA to enter into RBC and bind to its receptor, ABA transport across the plasma membrane is necessary.…”