Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.