Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import.Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145-and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit' peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized famtly of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit 18 of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.Chloroplast biogenesis depends on the import of a large number of diverse proteins that are synthesized in the cytoplasm as precursors with N-terminal transit peptides. A transit peptide mediates precursor recognition by receptors on the chloroplast envelope (1, 2), and upon membrane translocation into the stroma, it is proteolytically removed, yielding the mature protein (3, 4). Studies have indicated that a general stromal processing peptidase (SPP) with the properties of a metalloprotease cleaves the transit peptides of proteins destined for different functional complexes (3,4). Proteins targeted to the thylakoid lumen have a bipartite transit peptide that is cleaved first by SPP, then by a thylakoid protease (5-7). SPP thus plays a key role as part of the chloroplast import machinery.We have identified a soluble chloroplast processing enzyme (CPE) with characteristics of SPP that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein (LHCP) (8, 9). Antigenically related proteins of 145 and 143 kDa copurify with this activity from pea. Immunodepletion experiments show that the 145/143-kDa doublet is indeed required for cleavage of the LHCP precursor (preLHCP) and indicate that these proteins are involved in the removal of the transit peptides of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the acyl carrier protein.

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Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.

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