The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

A series of kinetic isotope effect experiments were performed with the goal of understanding the nature of rate-limiting steps in the soybean lipoxygenase-1 (SBL-1) reaction. SBL-1 was reacted with linoleic acid (LA) and deuterated linoleic acid (D-LA) under a variety of experimental conditions involving changes in temperature, pH, viscosity, and replacement of H2O with D2O. The extrapolated intrinsic primary H/D isotope effect can be estimated to be possibly as large as 80. This value is probably the largest isotope effect published for an enzymatic reaction, and much larger than that predicted from semiclassical models. Due to this large primary isotope effect, the C-D bond cleavage fully limits the rate of reaction under all conditions tested. In the case of protonated linoleic acid, a number of steps are partially rate-limiting at room temperature; three distinct mechanistic steps which include substrate binding, an H2O/D2O sensitive step, and C-H bond cleavage have been characterized. Use of glucose as a solvent viscosogen demonstrates that substrate binding is approximately 48% rate-limiting for LA at 20 degrees C. SBL-1 is one of the few enzymes that fit the definition of a "perfect enzyme", in the sense that further optimization of any single step at room temperature will not significantly increase the overall rate. At lower temperatures, the step sensitive to solvent deuteration begins to dominate the reaction, whereas at higher temperatures, the hydrogen abstraction step is rate-limiting. The pH dependence of kcat/Km for SBL-1 can be explained as arising from two pKa's, one controlling substrate binding and the other substrate release. Below pH 7.8, the rate of substrate release increases, thus decreasing the commitment to catalysis and unmasking the large intrinsic isotope effect on the subsequent hydrogen abstraction. An abnormally high pKa, in the range of 7-8, has been determined for LA in the concentration range employed in these studies. We propose that the negatively charged form of LA, predominating above pH 7.8, is the preferred substrate with larger commitments to catalysis.

SUMMARY Mitochondria play central roles in integrating pro- and anti-apoptotic stimuli and JNK is well-known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a non-phosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.

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