Mitochondrial division in Caenorhabditis elegans.

The study of mitochondrial division proteins has largely focused on yeast and mammalian cells. We describe methods to use Caenorhabditis elegans as an alternative model for studying mitochondrial division, taking advantage of the many wonderful resources provided by the C. elegans community. Our methods are largely based on manipulation of gene expression using classic and molecular genetic techniques combined with fluorescence microscopy. Some biochemical methods are also included. As antibodies become available, these biochemical methods are likely to become more sophisticated.

Mitochondrial localization of antizyme is determined by context-dependent alternative utilization of two AUG initiation codons.

Ornithine decarboxylase-antizyme (Az), a polyamine-induced protein that targets ornithine decarboxylase (ODC) to rapid degradation, is synthesized as two isoforms. Studies performed in vitro indicated that the 29 and 24.5 kDa isoforms originate from translation initiation at two alternative initiation codons. Using transient transfections we demonstrate here that also in cells the two isoforms are synthesized from two AUG codons with the second being utilized more efficiently. The more efficient utilization of the second AUG is due to its location within a better sequence context for translation initiation. By using immunostaining we demonstrate that only the less expressed long form of Az is localized in the mitochondria. Moreover, this long isoform of Az and not the more efficiently expressed short isoform is imported into mitochondria in an in vitro uptake assay. Our data therefore demonstrate that a single Az transcript gives rise to two Az proteins with different N-terminal sequence and that the longer Az form containing a potential N-terminal mitochondrial localization signal is transported to mitochondria.

Degradation of ornithine decarboxylase in Saccharomyces cerevisiae is ubiquitin independent.

Ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine biosynthesis is one of the most rapidly degraded proteins in eukaryotic cells. Mammalian ODC is a notable exception to the widely accepted dogma that ubiquitination is always required for targeting a protein to degradation by the 26S proteasome. However, while it is well established that in mammalian cells degradation of ODC is ubiquitin independent, the requirement of ubiquitination for degradation of ODC in yeast cells remained undetermined. We have investigated ODC degradation in three mutant strains of Saccharomyces cerevisiae in which ubiquitin-dependent protein degradation activity is severely compromised. While yeast ODC was rapidly degraded in all these mutant strains the degradation of N-end rule substrates was inhibited. A mutant mouse ODC that fails to interact with Az was rapidly degraded in yeast cells but was stable in mammalian cells suggesting that interaction with a mammalian Az like yeast protein is not necessary for the degradation of ODC in yeast cells. Deletion analysis revealed that sequences from its unique N-terminus are involved in targeting yeast ODC to rapid degradation in yeast cells.

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.