INTRACELLULAR-DISTRIBUTION OF STRESS GLYCOPROTEINS IN A HEAT-RESISTANT CELL MODEL EXPRESSING HUMAN HSP70

Heat stress results in the cellular accumulation of heat-shock protein s (HSP) and increased protein glycosylation. Among known stress glycop roteins, GP50 and GP62 are associated with the expression of thermotol erance. In the present study, we characterized subcellular localizatio n and redistribution of GP62 and GP50 in a rodent cell line, M21, befo re and after cellular heat-stress. M21 cells are heat-resistant cells that overexpress human HSP70 and also have concomitantly high GP62 lev els. Cellular fractionation by differential centrifugation showed that GP62 and GP50 was present in each subcellular fraction. However, each stress glycoprotein exhibited a characteristic kinetic pattern of red istribution during cellular recovery after heat stress. For example, g lycosylated GP62 was seen predominantly in the mitochondria before hea t-stress. Immediately after heat-stress, its presence in the mitochond rial fraction was dramatically reduced, while it increased in lysosome s, microsomes and cytosol. By 1 h after heat stress, it had largely di sappeared from microsomal and cytosolic fractions. After 24 h, all sub cellular fractions showed only trace amounts of residual GP62. By comp arison, GP50 was also highest in the mitochondrial fraction before hea t-stress, redistributed like GP62 immediately after heat stress, but r emained relatively unchanged thereafter. In contrast to GP62, GP50 sho wed little redistribution during 24 h after heat-stress and remained a t high concentrations in all cell fractions, including microsomes. Dis tribution of GP50 and GP62 before and after heat stress, based on diff erential centrifugation, was consistent with immunolocalization data. Following heat stress, both GP50 and GP62 showed a partial overlap in distribution with HSP70. The above results indicate that each stress g lycoprotein has a specific subcellular location, both before and after heat stress. The presence of GP62 in virtually all cell fractions is consistent with a multifunctional role for GP62 in the cellular stress response. (C) 1997 Academic Press.