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8.A.68 The Endomembrane protein-70 (EMP70) Family

The EMP70 family, also called the nonaspanin or Transmembrane 9 (TM9 or TMN) family, includes thousands of members with 9 or 10 TMSs with one TMS at the N-terminus and 9 more in a 1 + 2 + 2 + 2 + 2 TMS arrangement in the C-terminal halves of the proteins. The transmembrane domain is called the EMP70 domain. Homologues are found in all types of eukaryotes. The EMP70 proteins of plants and fungi are also called the nonaspanin domain superfamily (e.g., TM9SF2 or TM9SF4). TM9 proteins are localized in the secretory pathway of eukaryotic cells and are involved in cell adhesion and phagocytosis (Benghezal et al. 2003).

Hegelund et al. 2010 have investigated the physiological function of TM9 proteins. Cellular copper contents in Saccharomyces cerevisiae varied depending on the presence of TM9 homologues. The same is likely for Arabidopsis thaliana (Gao et al. 2012). A yeast tmn1-3 triple mutant lacking all three endogenous homologues (TMN1-3) showed altered metal homeostasis with a reduction in the cellular Cu contents to 25% of that in the wild-type. Conversely, when TMN1 was overexpressed in yeast, cellular Cu concentrations more than doubled. Both Tmn1p-GFP and Tmn2p-GFP fusion proteins localized to the tonoplast. Yeast vacuolar biogenesis was not affected by the lack or presence of TM9 proteins, neither in the tmn1-3 triple mutant nor in TM9 overexpressing strains. Heterologous expression in yeast of AtTMN7, a TM9 homologue from Arabidopsis, affected Cu homeostasis similar to the overexpression of TMN1. In Arabidopsis, the two TM9 homologues AtTMN1 and AtTMN7 were ubiquitously expressed. AtTMN7 promoter constructs driving the expression of GFP showed elevated expression of AtTMN7 in the root elongation zone. It was concluded that TM9 homologues from S. cerevisiae and A. thaliana have the ability to affect the intracellular Cu balance. It is likely that TM9 proteins are transporters of heavy metals, possibly catalyzing transport from the cytoplasm to the vacuolar lumen.

An inverted pH gradient across cell membranes is a typical feature of malignant cancer cells that are characterized by extracellular acidosis and cytosol alkalization. These dysregulations are able to create a unique milieu that favors tumor progression, metastasis and chemo/immune-resistance traits of solid tumors. A key event mediating tumor cell pH alterations is an aberrant activation of ion channels and proton pumps such as H+-vacuolar-ATPase (V-ATPase). TM9SF4 is related to cannibal behavior of metastatic melanoma cells (Bergeret et al. 2008). TM9SF4 is V-ATPase-associated and is involved in V-ATPase activation (Lozupone et al. 2009; Lozupone et al. 2015). In HCT116 and SW480 colon cancer cell lines, TM9SF4 interacts with the ATP6V1H subunit of the V-ATPase V1 sector. Suppression of TM9SF4 with small interfering RNAs strongly reduces assembly of V-ATPase V0/V1 sectors, thus reversing the tumor pH gradient with a decrease of cytosolic pH, alkalization of intracellular vesicles and a reduction of extracellular acidity. Such effects are associated with a significant inhibition of the invasive behavior of colon cancer cells and with an increased sensitivity to the cytotoxic effects of 5-fluorouracil.  A principle role of TM9 proteins is to serve as intramembrane cargo receptors, controlling exocytosis and surface localization of a subset of membrane proteins (Perrin et al. 2015). 

Nonaspanins (TM9SF) are characterized by an N-terminal TMS, followed by a large hydrophylic domain of about 180 aas, and nine putative transmembrane domains. There are 3 in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1-TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, and human TM9SF1 plays a role in the regulation of autophagy, and human TM9SF4 in tumor cannibalism. Pruvot et al. 2010 reported that the zebrafish genome encodes five members of this family, TM9SF1-TM9SF5. Expression analysis showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants and animals, revealed a conserved protein structure. An evolutionarily conserved immunoreceptor tyrosine-based inhibition motif was identifed in the cytoplasmic domain between TMS 7 and TMS 8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5. A conserved TRAF2 binding domain exists in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This suggests that TM9SF proteins play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity. Thus, these proteins, although possible heavy metal transporters, are auxilary subunits for other processes and are therefore retained in TC subclass 8.A. Some of these proteins may be homologous to ABC protein of TC subfamily 3.A.1.205.

Processes possibly mediated by EMP70 homologues are:

Copper ions (cytoplasm) ⟶ Copper ions (vacuole)

Regulation of the V-type ATPase

Exocytosis and surface localization of some membrane protein

References associated with 8.A.68 family:

Benghezal, M., S. Cornillon, L. Gebbie, L. Alibaud, F. Brückert, F. Letourneur, and P. Cosson. (2003). Synergistic control of cellular adhesion by transmembrane 9 proteins. Mol. Biol. Cell 14: 2890-2899. 12857872
Bergeret, E., J. Perrin, M. Williams, D. Grunwald, E. Engel, D. Thevenon, E. Taillebourg, F. Bruckert, P. Cosson, and M.O. Fauvarque. (2008). TM9SF4 is required for Drosophila cellular immunity via cell adhesion and phagocytosis. J Cell Sci 121: 3325-3334. 18796536
Bonache, S., F. Algaba, E. Franco, L. Bassas, and S. Larriba. (2014). Altered gene expression signature of early stages of the germ line supports the pre-meiotic origin of human spermatogenic failure. Andrology 2: 596-606. 24803180
Chen, L.J., N.N. Zhang, C.X. Zhou, Z.X. Yang, Y.R. Li, T. Zhang, C.R. Li, X. Wang, Y. Wang, Z.B. Wang, Z.R. Xia, Z.B. Wang, C.L. Zhang, Y.C. Guan, Q.Y. Sun, and D. Zhang. (2021). Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation. Cell Death Differ. [Epub: Ahead of Print] 34635817
Gao, C., C.K. Yu, S. Qu, M.W. San, K.Y. Li, S.W. Lo, and L. Jiang. (2012). The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus. Plant Cell 24: 2086-2104. 22570441
He, P., Z. Peng, Y. Luo, L. Wang, P. Yu, W. Deng, Y. An, T. Shi, and D. Ma. (2009). High-throughput functional screening for autophagy-related genes and identification of TM9SF1 as an autophagosome-inducing gene. Autophagy 5: 52-60. 19029833
Hegelund, J.N., T.P. Jahn, L. Baekgaard, M.G. Palmgren, and J.K. Schjoerring. (2010). Transmembrane nine proteins in yeast and Arabidopsis affect cellular metal contents without changing vacuolar morphology. Physiol Plant 140: 355-367. 20681974
Li, H., C. Li, X. Liu, and Z. Yang. (2022). A Golgi-Located Transmembrane Nine Protein Gene TMN11 Functions in Manganese/Cadmium Homeostasis and Regulates Growth and Seed Development in Rice. Int J Mol Sci 23:. 36555524
Lozupone, F., M. Borghi, F. Marzoli, T. Azzarito, P. Matarrese, E. Iessi, G. Venturi, S. Meschini, A. Canitano, R. Bona, A. Cara, and S. Fais. (2015). TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells. Oncogene 34: 5163-5174. 25659576
Lozupone, F., M. Perdicchio, D. Brambilla, M. Borghi, S. Meschini, S. Barca, M.L. Marino, M. Logozzi, C. Federici, E. Iessi, A. de Milito, and S. Fais. (2009). The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells. EMBO Rep 10: 1348-1354. 19893578
Paolillo, R., I. Spinello, M.T. Quaranta, L. Pasquini, E. Pelosi, F. Lo Coco, U. Testa, and C. Labbaye. (2015). Human TM9SF4 Is a New Gene Down-Regulated by Hypoxia and Involved in Cell Adhesion of Leukemic Cells. PLoS One 10: e0126968. 25961573
Perrin, J., M. Le Coadic, A. Vernay, M. Dias, N. Gopaldass, H. Ouertatani-Sakouhi, and P. Cosson. (2015). TM9 family proteins control surface targeting of glycine-rich transmembrane domains. J Cell Sci 128: 2269-2277. 25999474
Pruvot, B., V. Laurens, F. Salvadori, E. Solary, L. Pichon, and J. Chluba. (2010). Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish. Immunogenetics 62: 681-699. 20820770
Schimmöller, F., E. Díaz, B. Mühlbauer, and S.R. Pfeffer. (1998). Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution. Gene 216: 311-318. 9729438
Vernay, A., O. Lamrabet, J. Perrin, and P. Cosson. (2018). TM9SF4 levels determine sorting of transmembrane domains in the early secretory pathway. J Cell Sci 131:. 30301779