Characterizing functional differences in sea anemone Hsp70 isoforms using budding yeast

Abstract

Marine organisms experience abiotic stressors such as fluctuations in temperature, UV radiation, salinity, and oxygen concentration. Heat shock proteins (HSPs) assist in the response of cells to these stressors by refolding and maintaining the activity of damaged proteins. The well-conserved Hsp70 chaperone family is essential for cell viability as well as the response to stress. Organisms possess a variety of Hsp70 isoforms that differ slightly in amino acid sequence, yet very little is known about their functional relevance. In this study, we undertook analysis of three principal Hsp70 isoforms NvHsp70A, B, and D from the starlet sea anemone Nematostella vectensis. The functionality of Hsp70 isoforms in the starlet sea anemone was assessed through transcriptional analysis and by heterologous expression in budding yeast Saccharomyces cerevisiae. Interestingly, these isoforms were found to not only differ in expression under stress but also appear to have functional differences in their ability to mediate the cellular stress program. These results contribute to an understanding of Hsp70 isoform specificity, their shared and unique roles in response to acute and chronic environmental stress, and the potential basis of local adaptation in populations of N. vectensis.

Not quite the SSAme: unique roles for the yeast cytosolic Hsp70s

“…The starlet sea anemone Nematostella vectensis expresses 3 main cytosolic Hsp70 homologs, NvHsp70A, B and D. Expression of these isoforms parallels the expression patterns seen in yeast, where NvHsp70D is constitutively expressed in contrast to the stress-inducible NvHsp70A and B isoforms (Waller et al 2018). Given their high homology to Ssa1-4, it is unsurprising that NvHsp70A, B and D are capable of providing essential cell function in yeast lacking Ssa1-4 (Waller et al 2018). What is more interesting is that yeast solely expressing NvHsp70B from a constitutive promoter are generally more stress resistant than cells expressing the other Nematostella isoforms (Waller et al 2018).…”

“…Differential control of the heat shock response by specific Hsp70 isoforms has been observed in other species. The starlet sea anemone Nematostella vectensis expresses 3 main cytosolic Hsp70 homologs, NvHsp70A, B and D. Expression of these isoforms parallels the expression patterns seen in yeast, where NvHsp70D is constitutively expressed in contrast to the stress-inducible NvHsp70A and B isoforms (Waller et al 2018). Given their high homology to Ssa1-4, it is unsurprising that NvHsp70A, B and D are capable of providing essential cell function in yeast lacking Ssa1-4 (Waller et al 2018).…”

“…Given their high homology to Ssa1-4, it is unsurprising that NvHsp70A, B and D are capable of providing essential cell function in yeast lacking Ssa1-4 (Waller et al 2018). What is more interesting is that yeast solely expressing NvHsp70B from a constitutive promoter are generally more stress resistant than cells expressing the other Nematostella isoforms (Waller et al 2018).…”

Dynamic remodeling of the interactomes of Nematostella vectensis Hsp70 isoforms under heat shock

“…Yeast cultures were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) or in SD (0.67% yeast nitrogen base, 2% glucose) supplemented with the appropriate nutrients to select for plasmids and gene replacements. NvHsp70A, B, and D were amplified from cDNA synthesized from RNA isolated from Nematostella vectensis adults originally collected from Sippewissett, MA and inserted into the pAG415GPDccdB vector for expression in yeast using In-Fusion cloning as in [25]. All inserts were sequence confirmed with Sanger sequencing.…”

“…Transcriptome-wide surveys from adult N. vectensis cultured under various stressors have shown particular Hsps in each size class to have significant changes in expression [23,24]. Our previous studies demonstrate that when expressed as the sole Hsp70 in yeast, the NvHsp70 isoforms present unique cellular phenotypes suggesting they possess distinct biological functions [25].…”