A subset of lumen proteins is transported across the thylakoid membrane by a Sec-independent translocase that recognizes a twin-arginine motif in the targeting signal. A related system operates in bacteria, apparently for the export of redox cofactor-containing proteins. In this report we describe a key feature of this system, the ability to transport folded proteins. The thylakoidal system is able to transport dihydrofolate reductase (DHFR) when an appropriate signal is attached, and the transport efficiency is almost undiminished by the binding of folate analogs such as methotrexate that cause the protein to fold very tightly. The system is moreover able to transport DHFR into the lumen with methotrexate bound in the active site, demonstrating that the ⌬pH-driven transport of large, native structures is possible by this pathway. However, correct folding is not a prerequisite for transport. Truncated, malfolded DHFR can be translocated by this system, as can physiological substrates that are severely malfolded by the incorporation of amino acid analogs.

The biogenesis of the mitochondrial inner membrane is dependent on two distinct 70 kDa protein complexes. TIMM8a partners with TIMM13 in the mitochondrial intermembrane space to form a 70 kDa complex and facilitates the import of the inner membrane substrate TIMM23. We have identified a new class of substrates, citrin and aralar1, which are Ca2+-binding aspartate/glutamate carriers (AGCs) of the mitochondrial inner membrane, using cross-linking and immunoprecipitation assays in isolated mitochondria. The AGCs function in the aspartate-malate NADH shuttle that moves reducing equivalents from the cytosol to the mitochondrial matrix. Mohr-Tranebjaerg syndrome (MTS/DFN-1, deafness/dystonia syndrome) results from a mutation in deafness/dystonia protein 1/translocase of mitochondrial inner membrane 8a (DDP1/TIMM8a) and loss of the 70 kDa complex. A lymphoblast cell line derived from an MTS patient had decreased NADH levels and defects in mitochondrial protein import. Protein expression studies indicate that DDP1 and TIMM13 show non-uniform expression in mammals, and expression is prominent in the large neurons in the brain, which is in agreement with the expression pattern of aralar1. Thus, insufficient NADH shuttling, linked with changes in Ca2+ concentration, in sensitive cells of the central nervous system might contribute to the pathologic process associated with MTS.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec-and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified. ß