The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.Our earlier studies with mutants of Chinese hamster ovary (CHO) cells selected for resistance to the microtubule (MT) inhibitor podophyllotoxin showed that a large number of these mutants involved specific electrophoretic alteration in a major protein designated P1 (Mr, 63 kilodaltons [kDa]) (6, 7). The genetic lesion in these mutants appears to be related to the cellular action of the drug, since podophyllotoxinresistant mutants exhibit highly specific cross-resistance and collateral sensitivity to other MT inhibitors, such as colchicine, nocodazole, and taxol, and show reduced binding of the drug in cell extracts (6, 7). Immunofluorescence studies show that in interphase cells of vertebrate and invertebrate species, P1 antibody stains mitochondria, which show specific association with MTs (5, 7). Subfractionation of rat mitochondria has localized P1 to the matrix compartment (4). To help understand the cellular function of P1, cloning and sequencing of P1 cDNA from human cells was undertaken. The P1 sequence reported here shows extensive sequence and structural homology to a family of bacterial and plant proteins, termed chaperonins (8), which are involved in facilitating the posttranslational assembly of oligomeric protein complexes (1,3,8), as well as to the 65-kDa major antigenic protein of mycobacterial species (15,(18)(19)(20). The observed high degree of sequence and structural similarity between these proteins strongly indicates that P1 is the human homolog of this evolutionarily highly conserved group of proteins.Isolation of Pl-specific clones from kgtll libraries. We have previously demonstrated that our antibodies to P1 crossreact only with the P1 protein in one-and two-dimensional immunoblots of proteins from CHO and human cells (5, 7).

One of the mechanisms by which endothelial cells (ECs) regulate fibrinolysis is through the regulated assembly of proteins such as plasminogen, tissue plasminogen activator (tPA) and urokinase (uPA) on their membrane surface. Receptors for many of these fibrinolytic factors have been isolated and characterized. A unique 45 kD plasminogen receptor present on ECs derived from vein vasculature has been identified and resolved into two plasminogen binding components. One component consists of the unique 45 kD plasminogen receptor (pI = 6.3) whereas the other component (pI = 5.1) is identified as the cytoskeletal protein, actin. Immunofluorescent studies of isolated ECs confirm the presence of actin on their extracellular surface. This observation is consistent with a number of other recent reports of actin externally localized on other cell types. In vitro studies using purified actin confirm that plasminogen binds to actin both saturably and with relatively high affinity. Competition studies with lysine indicated that the binding was largely kringle-dependent, and when binding of tPA to actin was assessed, it also bound to actin with 70-80% of binding inhibited by lysine. Lipoprotein (a), which shows homology with plasminogen, also interacted with actin. Addition of plasminogen and low-density lipoproteins inhibited Lp(a) binding to actin in a dose-dependent fashion. Moreover, in competition with tPA, partial inhibition of plasminogen binding to actin was also observed. In experiments using anti-actin antibodies added in excess to cultured ECs, binding of plasminogen was inhibited by 45%, tPA binding was inhibited by 46% and Lp(a) binding was reduced by 56%, confirming actin as a binding site for these various ligands whilst attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing binding not only of plasminogen, but also of tissue plasminogen activator and Lp(a) to the surface of human endothelial cells.

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