Partial cloning of a squid microtubule-associated protein (MAP H1) and the identification of the microtubule binding domain.

An approximately 420-kDa ATP binding protein, referred to as MAP H1, has previously been shown to be involved in microtubule-dependent vesicle motility in the squid giant axon. To gain further insight into the structure and function of this protein, partially overlapping cDNA clones encoding approximately a quarter of the MAP H1 molecule were identified from two squid optic lobe libraries using affinity-purified antibodies to squid MAP H1. One clone in particular (KS18), which hybridizes to an approximately 13-kb message, encodes a series of almost identical repeats of a 16-amino-acid sequence that is tandemly repeated. The sequence of clone KS18 is unique and does not correspond to any nucleotide or amino acid sequence in the data base. The presence of repeated elements within the microtubule binding domain of several other MAPs prompted us to investigate whether the MAP H1 repeats are involved in microtubule binding. In-vitro-synthesized polypeptides containing these repeats sediment with taxol-stabilized microtubules in a microtubule binding assay. The predicted secondary structure of the 16-amino-acid repeat region of MAP H1 contains alternating beta-sheets and turns and could form the globular domain seen in negative-stain electron micrographs of MAP H1.

Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane.

The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.