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Characterization of solute carrier SLC 38 A 6

Transport across the membrane of a cell is of crucial importance for cellular functions. The solute carrier family, SLC38 is a family of membrane proteins that transports various substances through the membrane and thus performs many physiologically important functions, for example, transport of glutamine from astrocyte to neurons in the central nervous system. In this paper, we demonstrate that one of the transporters in this family named SLC38A6 forms several protein complexes with a variety of proteins in the membrane and in synaptic vesicles, suggesting that SLC38A6 is involved in the synaptic release of neurotransmitters in synapses. We performed sensitive protein interaction analysis between the protein of interest and a variety of proteins expressed at different sites in the neuronal cell. We showed that SLC38A6 interacts with proteins in the cell membrane as well as in the membrane of synaptic vesicles. The current theory is that SLC38A6 interact with these proteins when the synaptic vesicles are in close proximity with the cell membrane during the release of the neurotransmitters. Introduction The boundaries of cells as well as cell organelles are defined by their cell membrane. Approximately one third of the proteins from any organism are embedded in the cell membranes. These membrane proteins perform different cellular functions such as transport, enzymatic activity, signal transduction, cell to cell recognition, intercellular attachment and anchoring to the cytoskeleton. 30% of the proteins in the human genome are membrane proteins (Wu et al. 2003). It is of importance that the amino acid transport across the cell membrane as well as the mitochondrial and vesicular membranes is well-regulated (Hagglund et al. 2011). The solute Carriers (SLC:s) are the second biggest family of membrane proteins (Sundberg et al. 2008). These proteins transport a big range of substances over the cell membrane. Some of these substances are sugar, amino acids, nucleotides and inorganic ions (Hediger et al. 2004). There are many different families of solute carriers identified today and one of the interesting amino acid transporting solute carrier family is the SLC38 family. One of the major physiological role of SLC38 family transporters is to transport glutamine from astrocytes to neurons (Mackenzie and Erickson 2004). The SLC38 family consists of eleven proteins and five of them (SLC38A7-SLC38A11) have been identified in our group (Sundberg et al. 2008). The solute carrier family-38 is sodium-coupled neutral amino acid transporters (SNATs). Five of these proteins SLC38A1-SLC38A5 are well-studied; whereas, the rest of the six are still orphans. Amino acid transporter can be classified to system A and system N amino acid transporters depending on their functional properties and patterns of substrate recognition. The six amino acid transporters SLC38A6 to SLC38A11 are not yet classified according to the N/A system. The orphan transporters SLC38A7-SLC38A11 have relatively low sequence similarities in comparison with the characterized transporters. The expression pattern of the orphan transporters is unknown and is of importance for our understanding of functions for these transporters (Mackenzie and Erickson 2004, Hagglund et al. 2011). SLC38A1 and SLC38A2 are found to be expressed only in neurons. There is a lack of the protein SLC38A3 in the neurons in contrast to SLC38A1 and SLC38A2. SLC38A3 can be found in the brain, more specifically in astrocytes. SLC38A4 is expressed in liver and in placenta cells. The last protein in the SLC38 family whose expression pattern is well characterized is the SLC38A5. This transporter is expressed in brain, liver, kidney, lung, colon, small intestine, stomach, and spleen. SLC38A6 is an orphan transporter with not so well characterized expression patterns. However, expressed sequence tags have been found in placenta, spleen, muscle, brain and lungs (Sundberg et al. 2008). The transporter was detected in all tissue of mouse, with high levels in olfactory bulb and hypothalamus (Hagglund, unpublished). In this study we used in situ proximity ligation assay (PLA) to identify interaction patterns of SLC38A6. We have shown that SLC38A6 interact with various neuronal proteins to a different extent. SLC38A6 was found to interact with vesicular proteins, membrane proteins and ubiquitously expressed proteins in the synapses. The specificity and purity for SLC38A6 was attempted to verify by western blot analysis. Immunocytochemistry was done on the proteins to verify their expression patterns in the cells and to optimize the concentration of antibodies for PLA.