Substrate discrimination in the ubiquitin-proteasome system is believed to be dictated by specific combinations of ubiquitin-protein ligases (E3s) and ubiquitin-conjugating enzymes (E2s). Here we identify Doa10/Ssm4 as a yeast E3 that is embedded in the endoplasmic reticulum (ER)/nuclear envelope yet can target the soluble transcription factor Mat␣2. Doa10 contains an unusual RING finger, which has ubiquitin-ligase activity in vitro and is essential in vivo for degradation of ␣2 via its Deg1 degradation signal. Doa10 functions with two E2s, Ubc6 and Ubc7, to ubiquitinate Deg1-bearing substrates, and it is also required for the degradation of at least one ER membrane protein. Interestingly, different short-lived ER proteins show distinct requirements for Doa10 and another ER-localized E3, Hrd1. Nevertheless, the two E3s overlap in function: A doa10⌬ hrd1⌬ mutant is far more sensitive to cadmium relative to either single mutant and displays strong constitutive induction of the unfolded protein response; this suggests a role for both E3s in eliminating aberrant ER proteins. The likely human ortholog of DOA10 is in the cri-du-chat syndrome critical region on chromosome 5p, suggesting that defective ubiquitin ligation might contribute to this common genetic disorder.[Key Words: Ubiquitin; ERAD; proteasome; protein degradation; UPR] Selective protein degradation plays an essential role in a diverse array of biological processes. The most common mechanism for degrading intracellular proteins in eukaryotes uses the ubiquitin-proteasome system. In this system, polymers of ubiquitin, a 76-residue protein, are conjugated to a substrate protein, resulting in recognition and destruction of the substrate by the 26S proteasome (Hochstrasser 1996;Pickart 2001;Weissman 2001). For ubiquitin-protein conjugation, the C-terminal carboxyl group of ubiquitin is first activated in an energydependent reaction by the ubiquitin-activating enzyme (E1), followed by transfer of the ubiquitin to a ubiquitinconjugating enzyme (E2) via transthiolation. The E2, together with a third factor called a ubiquitin-protein ligase or E3, transfers ubiquitin to a lysine side-chain(s) of a target protein. E3s are factors that stimulate the E2-dependent ubiquitination of substrates (Reiss et al. 1989).The E3 proteins are thought to be largely responsible for the high degree of specificity in protein ubiquitination. For instance, the Rsp5 E3 enzyme ubiquitinates the large subunit of RNA polymerase II, and WW domains in Rsp5 interact directly with a repeated proline-rich motif (PxY) in the polymerase subunit (Chang et al. 2000). Whereas E1 and E2 components of the ubiquitin conjugation machinery can be readily identified by their signature sequence motifs, E3s have not been as easily categorized. However, known E3s divide into two heterogeneous families (Weissman 2001). E3s of the HECT domain family have a ∼350-residue domain that includes a conserved Cys residue, which attacks the ubiquitin-E2 to form another thioester intermediate before ubiquitin transfer...

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Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes ω-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.