The present results demonstrate that purified HAS reconstituted into liposomes mediates luminal CB efflux in a manner consistent with the presence of a HAS pore. HAS-mediated dye efflux was time-dependent and HAS concentration-dependent, and efflux did not occur or was greatly impaired if the enzyme was first inactivated by heat treatment or Cys -SH group modification by either oxidation or reaction with NEM. These results indicate that active native enzyme is required for efflux. HAS enzyme function is greatly affected by whether sufficient activating phospholipid is present. We previously found that purified SeHAS retains enough tightly bound endogenous lipid to retain low activity (and not be denatured), but the enzyme can be activated ≥ 10-fold by addition of TO-CL, containing C18:1 oleic acid chains; the best activating lipid identified so far [22
]. In contrast, TM-CL, containing C14:0 saturated fatty acids, does not activate SeHAS. The finding that HAS-mediated dye efflux was greater in liposomes with TO-CL compared to BCL and greater with BCL versus TM-CL is consistent with the presence of a pore whose size is dependent on the associated CL species.
An intraprotein pore in HAS-CL complexes may be smaller or distorted (thus slowing the rate of efflux) in the presence of the "bad" TM-CL species. Unlike other phosholipids, CL (also called diphosphatidylglycerol) has a "double" head group and four fatty acyl chains. Consequently, whereas other lipids occupy roughly cylindrical volumes, CL is shaped more like a cone with the narrower head group at the top. This structure imparts a natural tendency for CL to create or to localize to areas of negative (i.e
. concave) membrane curvature [28
]. We conclude that HAS could be activated by TO-CL, which favors negative curvature in the inner leaflet, because it would decrease lateral pressure on the protein and thus open up the cytoplasmic active sites and intraHAS pore [29
]. In contrast, HAS inhibition by TM-CL, whose shorter unkinked acyl chains favor a more cylindrical shape and more positive membrane curvature, could occur because it increases lateral pressure on the protein and would narrow the pore.
Since cloning and characterizing the first HAS [30
], we have been interested in understanding how HAS regulates the size of HA chains it assembles and how HA·HAS complexes can stay together for hours without dissociating during continuous polymerization of a growing chain at the reducing end (unlike the vast majority of glycosyltransferases that act at the nonreducing end). The characteristics of the HAS protein itself and how it functions led us to propose the Pore model for HA translocation through and by a HAS-lipid complex [1
]. For example, the lipid-dependence and 6-8 MDs of membrane-bound HAS enzymes are characteristics common to pore forming proteins [19
]. Additionally, the substrate binding sites are on the cytoplasmic side of the protein close to the cytoplasm-membrane interface [2
] and the enzyme is highly processive [15
Processive biosynthesis requires that after each catalytic cycle the HA-UDP product must transiently dissociate, without being released to diffusion, move relative to the enzyme active sites, and then rebind to HAS for the next cycle of sugar additions. As with other polymerases, the binding interaction between enzyme and polymer substrate cannot be of such high affinity that the rate of release, and thus rebinding of polymer, is too slow. Many RNA and DNA enzymes (e.g. polymerases and topoisomerases) have evolved solutions to this dilemma by creating topological or spatial constraints so that enzyme and product cannot dissociate. Molecular tethering strategies include formation of either multi-protein complexes that surround and encase the polymeric substrate or an intra-enzyme channel or pore to achieve a similar entrapment of the polymer.
This processivity of HA·HAS complexes is most consistent with the Pore model, which provides a topological mechanism to prevent dissociation during HA elongation. Since the 6-8 MDs of HAS enzymes are associated with multiple lipid molecules, it seems most likely that they utilize an intraHAS pore or deep cleft within the membrane that simultaneously serves to topologically constrain and also to provide a translocation mechanism to move the growing chain to the cell exterior. When dissociation does occur, HA release appears to terminate further elongation, perhaps due to loss of UDP at the reducing end - a chain terminating event. Even high affinity interactions (e.g. between receptors and ligands) typically have nM Kd values and measurable off-rates. The extremely slow, essentially immeasurable, off-rate for HA·HAS complex dissociation is inconsistent with enzyme reactions that use soluble substrates, which typically have Km values in the μM range, as proposed in the ABC transport model.
We previously identified Lys48
in MD2 and MD4, respectively, as important residues for the ability of SeHAS to produce large MDa HA [27
]; the K48E and K48F mutants make much smaller HA than WT. The interpretation that these two residues are within the proposed HAS pore and may interact with each other or with the growing HA chain during translocation is supported by the present findings that CB efflux mediated by both proteins is decreased compared to WT.
Since the present evidence supports the presence of an intraHAS pore through which HA could be translocated across membranes, it is reasonable to consider alternative interpretations that could account for reduced HA synthesis and secretion by inhibition of ABC transporters, such as MRP5. Two other explanations for decreased HA synthesis and secretion after inhibition or knockdown of MRP5 and other ABC transporters are related to the ability of these proteins to transport nucleotides (e.g
. cGMP, cAMP) as well as nucleotide-based drugs such as 5'-Fluoro-UMP, a metabolite of the anticancer drug 5'-Fluorouracil [37
]. The first possibility is that HAS inhibition could be caused by perturbation of the normal cellular metabolism of uracil-containing nucleotides, resulting in altered UDP-sugar concentrations or ability of precursor pathways to sustain the metabolic flux needed to make large amounts of HA. In eukaryotes, UDP-sugars are synthesized in the cytoplasm and levels are controlled by several mechanisms, including the Golgi anti-port system in which a UDP-sugar is transported in, while UMP is transported out. UMP deficiency causes the metabolic disorder orotic aciduria, but excess UMP (which leads to excess UDP) would also have a detrimental effect on HA synthesis. UDP is a potent substrate inhibitor of all Class I HASs [1
] and, in fact, can be used to quench HA synthase activity [15
]. Thus, deficiency or inhibition of MRP5 could lead to accumulation of cytoplasmic UDP and inhibition of HAS, an indirect result of MRP5 function unrelated to HA translocation. Both of the above possible indirect effects on HAS activity would be reversed by lysing cells (i.e
. dilution and mass action effects) and performing assays with UDP-sugars added in vitro
. Although these and other alternate explanations remain to be tested, Thomas and Brown recently found that ABC transporters are not involved in HA export by cancer cells [38