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Journal of Peking University (Health Sciences) ›› 2020, Vol. 52 ›› Issue (2): 221-226. doi: 10.19723/j.issn.1671-167X.2020.02.005

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Subcellular localization of GTPase of immunity-associated protein 2

Hong-quan QIN1,2,You ZHENG2,Man-na WANG2,Zheng-rong ZHANG2,Zu-biao NIU2,Li MA1,Qiang SUN2,Hong-yan Huang3,△(),Xiao-ning WANG1,△()   

  1. 1. School of Laboratory Medicine and Biotechnology, Institute of Molecular Immunology,Southern Medical University, Guangzhou 510515, China
    2. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing100071, China
    3. Department of Glioma, Beijing Shijitan Hospital, Capital Medical University, Beijing100038, China
  • Received:2019-12-16 Online:2020-04-18 Published:2020-04-18
  • Contact: Hong-yan Huang,Xiao-ning WANG E-mail:hhongy1999@126.com;xnwang88@163.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(81872314);Supported by the National Natural Science Foundation of China(31671432);the National Key Research & Development Program of China(2016YFC1303303);the National Key Research & Development Program of China(2018YFA0900804)

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Abstract:

Objective: To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study.Methods: In the study,we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM.Results: Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids,followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells.Conclusion: GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.

Key words: GTPase of immunity-associated protein 2, Bioinformatics, Subcellular localization

CLC Number: 

  • R392-33

Figure 1

The bioinformatics analysis of GIMAP2 proteinA, protein sequence of GIMAP2, yellow parts represent its transmembrane regions and red part represents the amino acids of nuclear export signal(NES); B, prediction of GIMAP2 transmembrane regions; C, prediction of GIMAP2 NES, transmembrane possibility(B) or NES score(C) on the vertical Y-axis against sequence position on the horizontal X-axis."

Figure 2

The construction process of pQCXIP-GIMAP2-mCherry recombinant vectorA, schema graph of pQCXIP-GIMAP2-mCherry recombinant vector; B, electrophoresis results of GIMAP2 gene fragment amplified by PCR; C, electrophoresis results of bacterial colony PCR; M, DNA marker Ⅲ; F, 1-6, PCR amplification products; D, sequencing result of pQCXIP-GIMAP2-mCherry recombinant vector."

Figure 3

Endogenous and exogenous GIMAP2 protein are co-localized in MDA-MB-436 cellA, C-terminally mCherry-tagged GIMAP2 (red) and endogenous GIMAP2 (green) are co-localized in MDA-MB-436 cells, nuclei was stained with DAPI, white arrow indicates gray scan position; B, green and red curves are the gray scan results of the corresponding fluorescence channel, vertical Y-axis represents fluorescence intensity while the horizontal X-axis represents white arrow's distance."

Figure 4

GIMAP2-mCherry fusion protein localizes to endoplasmic reticulum and lipid droplets Mito, specific marker dyes for mitochondria; ER, specific marker dyes for endoplasmic reticulum; BODIPY, specific marker dyes for lipid droplets."

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