Jens Preben Morth’s research group is focused on the structure and function of membrane proteins, and their interaction with lipids in the biological membrane.
Magnesium homeostasis in bacteria and plants
The biological significance of intracellular and extracellular magnesium is also linked to its transport across the membrane, and the regulation of this process. Thereby, the lipid bilayer is not passive, but generally functions as a structural framework for membrane components, and solvates membrane proteins.
However, given that eukaryote membranes are composed of numerous different lipid species, specific trafficking and clustering of lipids represents an additional level of functional regulation to embedded membrane proteins. Yet, the complexity of “the cellular lipidome” is still poorly understood. (Simons, and Sampaio, 2011; Vitrac, MacLean, et al, 2015).
Our recent work describes the first direct evidence that cardiolipin is colocalized in rafts with the magnesium transporter MgtA in the E. coli membrane and can activate MgtA in vitro. MgtA is a Magnesium transporting P-type ATPase activated at low Mg2+ levels peak when the molar ATP concentration is equivalent to the molar Mg2+total concentration.
Our data presents further evidence that free Mg2+ is the transported ion by MgtA, indicating that magnesium recognition likely takes place before magnesium enters the ion-binding site for occlusion (Subramani et al. 2016)) (see Figure 1).
As cardiolipin is present only in the mitochondria in plants and fungi, a still-unidentified lipid in the vacuolar membrane must be present for the MgtA homologs in plants and fungi. The crystal structures will allow us to understand the molecular function of magnesium transport, how they interact with cardiolipin, and how Mg2+, protons or co-factors are bound and recognized by this system. It will also enable us to start searching for the necessary co-factor in higher eukaryotes: at the moment, we can only search for structural homologs without knowing what aspect of the structure is important.
Structural and functional studies of the membrane bound E3 ubiquitin ligases
The ubiquitination system is a signalling system used to control a wide variety of cellular processes, in particular the proteolysis and degradation of other proteins. The system includes three enzymes 1, 2, and 3 (E1, E2, and E3). E1 is an ATPase that uses ATP to conjugate a ubiquitin molecule (Ub) to an E2. The now activated E2-Ub forms a protein complex with an E3 and a protein substrate to catalyse the transfer of Ub onto the protein substrate. The ubiquitinated protein thus signals to the cell that it has been tagged, for example, for degradation.
There are more than 600 E3 ligases in humans that control and target a broad range of cellular processes. Processes with dysfunctional regulation of E3 ligases are often linked to obesity, viral infections, neurodegenerative disorders, cancer, and stem cell regulation.
Two members of the membrane associated RING-CH (MARCH) family of E3-ligases, namely MARCH-1 and MARCH-8 (MARCH-1/8) are key players in the immune system and have been shown to regulate MHC-II antigen presentation by ubiquitination (Ohmura-Hoshino, Matsuki, et al, 2006; Shin, Ebersold, et al, 2006; Thibodeau, Bourgeois-Daigneault, and Lapointe, 2012; Walseng et al, 2010).
In addition, MARCH-1 was recently identified as a negative regulator of the insulin receptor signalling (Nagarajan, Petersen, et al, 2016) and MARCH-8 was shown to significantly reduce the infectivity of enveloped viruses, such as vesicular stomatitis virus and HIV (Tada, Zhang, et al, 2015). In contrast, the related mitochondrial MARCH-5 protein is involved in the maintenance of pluripotency in stem cells, and regulates mitochondrial morphology (Gu, Li, et al, 2015; Yonashiro, Ishido, et al, 2006). However, the molecular basis of membrane-bound E3 ligases and how they recruit their target proteins is still unknown.
We have recently reviewed the current knowledge on the membrane-imbedded MARCH E3 ligases (MARCH-1- 6, 8, 9, 11) with a focus on how the transmembrane regions can contribute via G/AxxxG/A-motifs to the selection and recognition of other membrane proteins as substrates for ubiquitination (Bauer, Bakke and Morth, 2016) (Figure 2). (G/AxxxG/A motifs are conserved regions in the protein sequence, with a glycine or alanine followed by three random amino acids and then, again, a glycine or alanine).
- Use MgtA as a benchmark system to understand molecular dynamics at the membrane protein-lipid interface important for activation or stability of membrane proteins.
- Understand how Mg2+ is specifically recognized by the MgtA-Cardiolipin complex and how an induced concentration difference of divalent ions could affect protein-lipid dynamics and structure.
- Use the membrane bound MARCH proteins to study protein-protein interaction taking place within a lipid bilayer.
- Understand how membrane proteins are targeted to their designed membrane in the cell, e.g. how MARCH-5 is targeted to the mitochondrial membrane while MARCH-1,8 are targeted to the endosomal membrane, or how MgtA is targeted to the poles and septal region in the bacterial plasma membrane.
- Understand the molecular parameters that govern target protein recognition of MARCH E3 ligases and to contribute to develop strategies for therapeutic regulation of MARCH-induced ubiquitination.
- Bauer, J., Bakke, O., and Morth J.P. (2016). Overview of the membrane-associated RING-CH (MARCH) E3 ligase family. N. Biotechnol. (Ahead of Print)
- Bourgeois-Daigneault, M.C., and Thibodeau, J. (2013). Identification of a novel motif that affects the conformation and activity of the MARCH1 E3 ubiquitin ligase. J. Cell. Sci. Pt 4, 989-998.
- Gu, H., Li, Q., Huang, S., Lu, W., Cheng, F., Gao, P., Wang, C., Miao, L., Mei, Y., and Wu, M. (2015). Mitochondrial E3 ligase March5 maintains stemness of mouse ES cells via suppression of ERK signalling. Nat. Commun. 7112.
- Nagarajan, A., Petersen, M.C., Nasiri, A.R., Butrico, G., Fung, A., Ruan, H.B., Kursawe, R., Caprio, S., Thibodeau, J., Bourgeois-Daigneault, M.C. et al. (2016). MARCH1 regulates insulin sensitivity by controlling cell surface insulin receptor levels. Nat. Commun. 12639.
- Nathan, J.A., and Lehner, P.J. (2009). The trafficking and regulation of membrane receptors by the RING-CH ubiquitin E3 ligases. Exp. Cell Res. 9, 1593-1600.
- Ohmura-Hoshino, M., Matsuki, Y., Aoki, M., Goto, E., Mito, M., Uematsu, M., Kakiuchi, T., Hotta, H., and Ishido, S. (2006). Inhibition of MHC class II expression and immune responses by c-MIR. Journal of Immunology 1, 341-354.
- Shin, J.S., Ebersold, M., Pypaert, M., Delamarre, L., Hartley, A., and Mellman, I. (2006). Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination. Nature 7115, 115-118.
- Simons, K., and Sampaio, J.L. (2011). Membrane organization and lipid rafts. Cold Spring Harb Perspect. Biol. 10, a004697.
- Subramani, S., Perdreau-Dahl, H., and Morth, J.P. (2016). The magnesium transporter A is activated by cardiolipin and is highly sensitive to free magnesium in vitro. Elife 10.7554/eLife.11407.
- Tada, T., Zhang, Y., Koyama, T., Tobiume, M., Tsunetsugu-Yokota, Y., Yamaoka, S., Fujita, H., and Tokunaga, K. (2015). MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins. Nat. Med. 12, 1502-1507.
- Thibodeau, J., Bourgeois-Daigneault, M.C., and Lapointe, R. (2012). Targeting the MHC Class II antigen presentation pathway in cancer immunotherapy. Oncoimmunology 6, 908-916.
- Vitrac, H., MacLean, D.M., Jayaraman, V., Bogdanov, M., and Dowhan, W. (2015). Dynamic membrane protein topological switching upon changes in phospholipid environment. Proc. Natl. Acad. Sci. U. S. A. 45, 13874-13879.
- Yonashiro, R., Ishido, S., Kyo, S., Fukuda, T., Goto, E., Matsuki, Y., Ohmura-Hoshino, M., Sada, K., Hotta, H., Yamamura, H., Inatome, R., and Yanagi, S. (2006). A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J. 15, 3618-3626.
- Walseng E., Furuta K., Bosch B., Weih K.A., Matsuki Y., Bakke O., Ishido S., Roche P.A. (2010) Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells, Proc. Natl. Acad. Sci. U. S. A. 107, 20465-20470.