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. 2016 Oct 10;17(1):792.
doi: 10.1186/s12864-016-3145-9.

Testis-Specific GTPase (TSG): An oligomeric protein

Sudeep Kumar  1 , Hyun Joo Lee  2 , Hee-Sae Park  1 , Keesook Lee  3
Affiliations

Testis-Specific GTPase (TSG): An oligomeric protein

Sudeep Kumar et al. BMC Genomics. .

Abstract

Background: Ras-related proteins in brain (Rab)-family proteins are key members of the membrane trafficking pathway in cells. In addition, these proteins have been identified to have diverse functions such as cross-talking with different kinases and playing a role in cellular signaling. However, only a few Rab proteins have been found to have a role in male germ cell development. The most notable functions of this process are performed by numerous testis-specific and/or germ cell-specific genes. Here, we describe a new Rab protein that is specifically expressed in male germ cells, having GTPase activity.

Results: Testis-specific GTPase (TSG) is a male-specific protein that is highly expressed in the testis. It has an ORF of 1593 base pairs encoding a protein of 530 amino acids. This protein appears in testicular cells approximately 24 days postpartum and is maintained thereafter. Immunohistochemistry of testicular sections indicates localized expression in germ cells, particularly elongating spermatids. TSG has a bipartite nuclear localization signal that targets the protein to the nucleus. The C-terminal region of TSG contains the characteristic domain of small Rab GTPases, which imparts GTPase activity. At the N-terminal region, it has a coiled-coil motif that confers self-interaction properties to the protein and allows it to appear as an oligomer in the testis.

Conclusion: TSG, being expressed in the male gonad in a developmental stage-specific manner, may have a role in male germ cell development. Further investigation of TSG function in vivo may provide new clues for uncovering the secrets of spermatogenesis.

Keywords: GTPase; RASEF; Testis.

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Figures

Fig. 1
Fig. 1
Exonic organization of Rasef and Tsg. The size of boxes corresponds to the size of exons, while the space between the boxes corresponds the introns for the genomic organization of predicted mouse Rasef (top) and Tsg (bottom) (a) Representation of the first two exons of Tsg in comparison with the first four exons of Rasef. The in-frame upstream stop codon and the start codon for Tsg are underlined with single and double lines, respectively (b)
Fig. 2
Fig. 2
Expression analysis of TSG in mouse tissues and developing testis. Total RNAs used in northern blot analysis were isolated from various tissues of adult mice and 28S rRNA was used as a loading control (a) Expression of Tsg is specific to the testis as analyzed in different tissues of 6-week-old mice by RT-PCR. β-actin was used as an endogenous control (b) Total RNAs from the indicated age of developing testis were used for the analysis (c) Expressional analysis of Tsg in the indicated aged testis by RT-PCR (d)
Fig. 3
Fig. 3
Expression of TSG protein in mouse tissues and developing testis. TSG exists as a higher molecular weight form in the testis (a) In vitro translation using radioactively labelled methionine clearly shows two forms of TSG produced from the cDNA clone after autoradiography. Similar forms of TSG were detected using anti-TSG antibody in immunoblot analyses using the lysates of 293T cells transfected with HA-tagged TSG. (‡) stands for samples of in vitro translation using radioactive methionine. Empty pCDNA HA vector was used as a negative control (b) TSG expression during testis development (c)
Fig. 4
Fig. 4
TSG is highly expressed in elongating spermatids. Immunofluorescence of the adult testis sections of mice showing stage VII tubule with Alexa 488 anti-TSG antibody (GREEN; left), TO-PRO-3 (RED, middle) and merge (right). Inset represents a higher magnification
Fig. 5
Fig. 5
Structural organization of TSG protein. The N-terminal region harbors the coiled-coil motif and the C-terminus contains the Rab domain. Bipartite nuclear targeting sequence is located in the middle of coiled-coil motif. The coiled-coil region also harbors the TPR/MLP1/MLP2-like domain
Fig. 6
Fig. 6
TSG is able to self-interact through the coiled-coil motif. Immunoprecipitation using anti-HA antibody (a) or anti-FLAG antibody (b) The immunoprecipitates were analyzed by western blotting using anti-FLAG and anti-HA antibodies. All HA- and FLAG-tagged constructs were cloned into the pCDNA3 vector, and 5 % of the total protein was taken as input. The asterisk (*) represents the non-specific IgG
Fig. 7
Fig. 7
TSG is likely a nuclear protein. GFP-tagged TSG expressed exogenously in HeLa cells was concentrated predominantly in the nucleus (Green) while N-terminus-deleted TSG was localized to the cytoplasm similar to GFP (Green) only. Nuclear DNA was stained using TO-PRO-3 (Red)
Fig. 8
Fig. 8
Functional characteristics of TSG. TSG has GTPase activity. The extract from Cos-7 cells transfected with pEGFP or pEGFP-TSG was incubated with γ- [32P]-GTP for 24 h and then subjected to TLC (a) TSG binds to actin inside the cell. Cos-7 cells were transfected with GFP only or with GFP-TSG, and lysates were immunoprecipitated with anti-GFP antibody. A total of 10 % of the sample was loaded as input (b)
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