ANKRD22 is an N-myristoylated hairpin-like monotopic membrane protein specifically localized to lipid droplets

Toshihiko Utsumi,Motoaki Otsuka,Aya Kiwado,Manami Yano,Misato Nishiue,Natsuko Iwamoto,Mayu Shichita,Takuro Hosokawa,Haruna Harada,Koko Moriya

doi:10.1038/s41598-021-98486-8

Abstract

The membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined. Immunofluorescence staining of COS-1 cells transfected with cDNA encoding ANKRD22 coupled with organelle markers revealed that ANKRD22 localized specifically to lipid droplets (LD). Analysis of the intracellular localization of ANKRD22 mutants C-terminally fused to glycosylatable tumor necrosis factor (GLCTNF) and assessment of their susceptibility to protein N-glycosylation revealed that ANKRD22 is synthesized on the endoplasmic reticulum (ER) membrane as an N-myristoylated hairpin-like monotopic membrane protein with the amino- and carboxyl termini facing the cytoplasm and then sorted to LD. Pro98 located at the center of the predicted membrane domain was found to be essential for the formation of the hairpin-like monotopic topology of ANKRD22. Moreover, the hairpin-like monotopic topology, and positively charged residues located near the C-terminus were demonstrated to be required for the sorting of ANKRD22 from ER to LD. Protein N-myristoylation was found to positively affect the LD localization. Thus, multiple factors, including hairpin-like monotopic membrane topology, C-terminal positively charged residues, and protein N-myristoylation cooperatively affected the intracellular targeting of ANKRD22 to LD.

Highlights

The membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined
In this study, protein N-myristoylation, membrane localization, intracellular localization, and membrane topology of ANKRD22 expressed in mammalian cells were examined
We investigated the role of the hairpin-like monotopic topology and protein N-myristoylation in the intracellular localization of ANKRD22 by immunofluorescence microscopic analysis using wild-type ANKRD22-glycosylatable tumor necrosis factor (GLCTNF) and two mutants, ANKRD22-P98A-GLCTNF and ANKRD22-G2A-GLCTNF

Summary

Introduction

The membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined. Multiple factors, including hairpin-like monotopic membrane topology, C-terminal positively charged residues, and protein N-myristoylation cooperatively affected the intracellular targeting of ANKRD22 to LD. In addition to mediating mitochondrial targeting, protein N-myristoylation plays an important role in the specific physiological functions of N-myristoylated membrane proteins. ANKRD22, a functionally unknown protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, was found to be an N-myristoylated hairpinlike monotopic membrane protein localized to lipid droplets (LD).

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Results

Conclusion