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

Abstract

A major cellular protein (P2; -70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors coichicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc7O). The inference that P2 and hsc7O are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc7O; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp7O with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp7O in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp7O antibodies in human and CHO cells; and (v) a cDNA clone for hsc7O has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc7O in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.All procaryotic and eucaryotic cells studied to date exhibit increased synthesis of a set of related proteins, referred to as heat shock proteins (hsps), in response to sudden increase in physiological growth temperature as well as other stresses (e.g., transition metals, amino acid analogs, teratogens, and mutagens) (16,17,23). In eucaryotic cells, the major hsp produced has an apparent subunit size of 70 kilodaltons (kDa) (hsp70) and it is highly conserved, even in evolutionarily distant relatives (17). Recent studies indicate that a multigene family encodes hsp70-related proteins, some of which are constitutively expressed and show little or no induction upon heat shock (7,17,22,26). The constitutive form of hsp70 (referred to as hsc70 in this article) has a pI of 5.8 and constitutes a major protein in unstressed rat and human cells (7,22). In contrast, a more basic form (pl, 5.9) of hsp70 is highly induced by heat shock or by other stress stimuli (14,17).Current evidence shows that the constitutive and inducible forms of hsp70 are encoded by different but homologous genes (16,17). 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...

Chaperones increase association of tau protein with microtubules

“…We wished to determine whether this association of tau with Hsp70͞90 occurs between free tau and chaperones or microtubule-associated tau and chaperones. We reasoned that it would be difficult to address this in cells by using immunofluorescence analysis because Hsp90͞70 are diffusely distributed in the cytosol; although it has been shown that Hsp70 also colocalizes with microtubules (34)(35)(36). Therefore we used a technique involving cell permeabilization and removal of cytosol leaving microtubules and some cytosolic constituents intact (Fig.…”

The active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics

“…Hsc70 associates with both intact MTs (15)(16)(17)(18)(19) and tubulin directly through its nucleotidebinding domain (NBD), suggesting functional regulation of tubulin by Hsc70 beyond proteostasis (20). Hsc70 also interacts with the microtubule (MT)-associated protein (MAP) tau, a putative 'typical' client of Hsc70 (6,(21)(22)(23) further suggesting that Hsc70 uniquely regulates MT dynamics (24,25). In addition to its role in MT stability, abnormal tau accumulation is linked to the pathology of a number of neurodegenerative diseases including Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease and chronic traumatic encephalopathy (26).…”

Cloning of the HSP70 gene from Halobacterium marismortui: relatedness of archaebacterial HSP70 to its eubacterial homologs and a model for the evolution of the HSP70 gene

“…Although the synthesis of HSP70 is greatly enhanced by various physiological stressors, it also constitutes a major protein under normal growth conditions and has been shown to be essential for cellular growth. Gene cloning and sequencing studies on HSP70 show that the primary structure of this protein has been highly conserved during evolution in species ranging from bacteria to plants to humans (27,34).In recent years, although HSP70 homologs have been cloned from numerous bacterial and eukaryotic species (1,2,4,6,12,14,18,21,32,37,38,41,42) …”

“…In recent years, although HSP70 homologs have been cloned from numerous bacterial and eukaryotic species (1,2,4,6,12,14,18,21,32,37,38,41,42) …”