In this work, TetL was shown to exist as an oligomer rather than a monomer in the membrane. The major piece of evidence came from tests of the expectation that if TetL exists as an oligomer in the membrane, some hetero-oligomers containing both His- and T7- tags would be found in membranes of cells expressing TetL proteins labeled with these two different tags ( and ). Consequently, we would be able to identify T7-tags in protein samples that were purified by His-tag directed Co2+-TALON affinity chromatography. Thus, the starting criterion was fulfilled for positing a TetL oligomeric state in the membrane (&).
Illustration showing dimeric organizations of cells or mixed membrane vesicles that overexpressed combinations of TetL-His and T7-TetL.
Possibilities of TetL oligomeric states in the membrane
Evidence for a dimeric TetL complex comes from a number of observations (). First, association was seen between differently tagged TetL subunits only when they were co-expressed in the same cell (&). When subunits in membranes of different cells were mixed prior to solubilization, no such interaction was observed, indicating that transporter complexes formed in the membrane and were stable enough to withstand detergent extraction from the membrane. This is very similar to the results obtained for NhaA (15
). Second, a TetL dimer band in denaturing PAGE experiments was seen during protein purification, even at low protein concentrations (), indicating a strong interaction of TetL subunits within the dimer. No higher oligomers were observed under these conditions. The behavior of TetL is thus similar to those of other stable oligomeric membrane proteins like KscA K+-channel (41
). Third, evidence for a TetL dimer was obtained from analytical size exclusion chromatography of purified protein, where purified TetL eluted as a dimer in detergent solution (,&). The existence of TetL dimmer in solution was observed across broad pH and temperature ranges, and in a variety of different detergents, making it unlikely to be an artifact caused by specific protein-detergent interactions.
We cannot rule out the possibility that higher oligomers, such as tetramers, exist in the membranes (). It is possible that tetramers in the membrane dissociated into dimers upon solubilization. Whereas the monomer-monomer interactions within a dimer are clearly very strong, dimer-dimer interactions, if any, must be relatively weak and thus less important for proper functioning of the protein. Given the prevalence of a dimer under diverse conditions in solution, we hypothesize that a dimer is the in vivo structure as well.
It remains unclear whether the dimeric association of TetL is necessary for substrate translocation. As noted, several MFS proteins have been shown to function as monomers, including Glut1 (18
), LacY (20
) and UhpT (22
). On the other hand, although the functional unit of LacS is a monomer, it forms a dimer in the membrane with two sugar translocation pathways that exhibit cooperative behavior (26
). Most importantly, the recently determined structures of glycerol-3-phosphate transporter and the lactose permease, both MFS proteins, reveal that the substrate translocation pore is located between the N
- and C
-terminal halves of the proteins (8
). Since all MFS proteins share a similar topology and even some degree of sequence homology, it is likely that they all work as monomers.
The complementation observed by Rubin and Levy with Gram-negative Tet proteins (39
) and additional properties have led to a proposal for TetB in which a dimeric Tet might be able to form a translocation pathway within a monomer but could also form a dimer in which two translocation pathways were formed through interactions between the two complementing domains of the protein (25
). This latter option probably does not apply to the larger TetL, inasmuch as complementation among mutations in different regions was not detected. Large-scale conformational changes between the two halves of monomeric GlpT are thought to be necessary for substrate translocation. In an MFS oligomer, such gross conformational changes are likely to affect the transport activity of the other subunit(s). This means that various subunits would be expected function cooperatively. Therefore, oligomerization, as observed with the TetL protein, provides another dimension of regulation, e.g. by allosteric interactions, which may be particularly important for this multifunctional antiporter. During earlier kinetic experiments (42
observed stimulation of tetracycline efflux by very low concentrations of a competitive efflux substrate, Na+; such stimulation at low concentrations and competitive inhibition at higher concentrations is most consistent with a cooperative interaction that would be best accommodated by an oligomeric transporter. Once the appropriate mutants of TetL are available for complementation experiments, it is anticipated that the physiologically-related functions of the TetL dimer will be shown to relate to elements of regulation, such as the different pH dependence of its different catalytic modes (1
), analogous to the findings with NhaA (15
). The challenge of demonstrating the functional implications of transporter oligomerization are not, however, trivial and there are only a few clear examples thus far (10