Intracellular distribution of stress glycoproteins in a heat-resistant cell model expressing human HSP70

Heat stress results in the cellular accumulation of heat-shock proteins (HSP) and increased protein glycosylation. Among known stress glycoproteins, GP50 and GP62 are associated with the expression of thermotolerance. In the present study, we characterized subcellular localization and redistribution of GP62 and GP50 in a rodent cell line, M21, before and after cellular heat-stress. M21 cells are heat-resistant cells that overexpress human HSP70 and also have concomitantly high GP62 levels. 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 redistribution during cellular recovery after heat stress. For example, glycosylated GP62 was seen predominantly in the mitochondria before heat-stress. Immediately after heat-stress, its presence in the mitochondrial fraction was dramatically reduced, while it increased in lysosomes, microsomes and cytosol. By 1 h after heat stress, it had largely disappeared from microsomal and cytosolic fractions. After 24 h, all subcellular fractions showed only trace amounts of residual GP62. By comparison, GP50 was also highest in the mitochondrial fraction before heat-stress, redistributed like GP62 immediately after heat stress, but remained relatively unchanged thereafter. In contrast to GP62, GP50 showed little redistribution during 24 h after heat-stress and remained at high concentrations in all cell fractions, including microsomes. Distribution of GP50 and GP62 before and after heat stress, based on differential 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 glycoprotein 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.