The Brain 68‐Kilodalton Microtubule‐Associated Protein Is a Cognate Form of the 70‐Kilodalton Mammalian Heat‐Shock Protein and Is Present as a Specific Isoform in Synaptosomal Membranes

Abstract

The relationship between the 68-kilodalton microtubule-associated protein (68KMAP) and the major heat-induced protein (HSP70) in rat and human cells was investigated by comparison of their heat induction properties and by tryptic and Cleveland peptide mapping procedures. HSP70 synthesis was induced by heat shock of rat and human cells, whereas 68KMAP was a major synthesised protein in the absence of heat shock, with its synthesis being only slightly increased on heat shock. Tryptic peptide mapping, however, indicated strong peptide homology between the two proteins. These data, therefore, confirm that 68KMAP represents a constitutively expressed, heat-shock cognate gene. Two-dimensional gel electrophoretic analysis of subcellular fractions of rat brain, combined with peptide mapping procedures, indicated that 68KMAP exists as at least two isoforms separable by isofocussing, the more acidic of which (alpha 68KMAP) is present in fractions enriched in microtubules, cytosol, microsomes, synaptosomal plasma membranes, and synaptic vesicles, and the more basic of which (beta 68KMAP) is present predominantly in fractions enriched in synaptic vesicles and synaptosomal plasma membranes. These two forms are distinguishable in terms of changes in Cleveland peptide maps, and we conclude that alpha- and beta 68KMAP, therefore, represent distinct forms. The significance of these findings to the molecular pathogenesis of Down's syndrome in the human brain is discussed.

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family.

“…In terms of their function, recent studies show that the hsp70 group of proteins perform an ATP-dependent protein-unfolding function which is involved in the disruption of protein complexes and in the transport of proteins into mitochondria and other organelles (5,6). In addition, there is considerable evidence indicating association of these proteins with microtubules (MTs) and other cytoskeletal structures (19)(20)(21)30), but their exact role in this regard is not clear at present.For the past few years we have used a combined genetic and biochemical approach to understand the structure and function of MTs in mammalian cells. Previous studies from our laboratory with Chinese hamster ovary (CHO) cell mutants resistant to the MT inhibitors podophyllotoxin and colchicine have identified two different proteins, P1 (-63 * Corresponding author.…”

An estradiol-induced protein synthesized in the ventral medial hypothalamus and transported to the midbrain central gray

“…These stress proteins are synthesized in neurons (Greenberg and Lasek, 1985) and axonally transported (Clark and Brown, 1985). The stress proteins are also similar-by peptide mapping, molecular weight, and PI-to another set of brain isoproteins, the microtubule associated proteins (MAPS) (Whatley et al, 1986). The 70 kDa (relatively basic) isoform of MAP whose p1 seems most similar to the E,-induced protein reported here is associated specifically with brain synaptic vesicles (Whatley et al, 1986).…”

“…The stress proteins are also similar-by peptide mapping, molecular weight, and PI-to another set of brain isoproteins, the microtubule associated proteins (MAPS) (Whatley et al, 1986). The 70 kDa (relatively basic) isoform of MAP whose p1 seems most similar to the E,-induced protein reported here is associated specifically with brain synaptic vesicles (Whatley et al, 1986). Since this (relatively basic) synaptic isoform of MAP is, by peptide mapping, very similar to the stress proteins, all ofwhich have clathrin binding activity in the presence of ATP (Ungewickell, 1985), this isoform of MAP may be the 5.9 p1 minor isoform of uncoating protein.…”

Members of the 70-kilodalton heat shock protein family contain a highly conserved calmodulin-binding domain.

“…Hsp7O proteins have also been found to associate with a number of cellular proteins. The identities of most of these proteins are unknown; however, hsp70 has been shown to bind to cytoskeletal elements, a cell surface glycoprotein, and calmodulin (7,16,31,33). Oligomeric protein complexes containing hsp70 and p53 tumor antigen have been isolated from transformed cells (15).…”