Abstract. The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.

We have studied the gifiace of the N-ter- were generated using the "gene splicing by overlap extension" method (13, 14). Sequences coding for watermelon pre-gMDH (2) and pre-mMDH (3) were inserted as 1.3-kb Not I-Sal I fragments in pGEM-5Zf(+) (Promega) and were used as template in PCR experiments. For construction ofthe gene fusions and the introduction of mutations in the preseAbbreviations: MDH, malate dehydrogenase; gMDH, glyoxysomal MDH; mMDH, mitochondrial MDH. §To whom reprint requests should be addressed

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons two hsps with molecular masses of 70 and 72 kDa. Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids. Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber. A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids. The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids in vitro. The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X 5 -KL) that directed in vivo a reporter protein into peroxisomes of the yeast Hansenula polymorpha. Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context. We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence.Seventy-kilodalton heat shock proteins (hsp70s) serve as ubiquitous molecular chaperones in DNA replication, protein folding, and transport. Organelle hsp70s (e.g., in plastids, mitochondria, and endoplasmic reticulum) characteristically contain N-terminal presequences with the organelle-specific targeting information; the endoplasmic reticulum hsp70 is also provided with a Cterminal retention signal. In eukaryotic cells cytosolic hsp70s play an essential role in the delivery of proteins to their target organelle by binding to nascent polypeptide chains and thereby keeping them in a translocation competent conformation. Organellar hsp70s located, for example, in the plastid or mitochondrial matrix and in the endoplasmic reticulum lumen, are involved in the protein translocation process (1-3). So far, hsp70s have not been encountered in microbodies (peroxisomes͞glyoxysomes), though cytosolic hsp70s have been implicated to stimulate peroxisomal protein import in in vitro studies with permeabilized cells (4) and are associated with the outside of the microbody membrane (5,26). Their specific in vivo role is still a matter of debate since the peroxisomal protein import machinery seems capable of transferring oligomeric folded proteins (6).In this paper, we describe the isolation of a watermelon (Citrullus vulgaris) hsp70 that occurs in its mature form in both plastids and glyoxysomes. Analysis of mRNA molecules for this hsp70 reveals two start methionine in frame within the presequence leading to two type...

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.