The C-terminal extension of Saccharomyces cerevisiae Hsp104 plays a role in oligomer assembly.
The Saccharomyces cerevisiae protein Hsp104, a member of the Hsp100/Clp AAA+ family of ATPases, and its orthologues in plants (Hsp101) and bacteria (ClpB) function to disaggregate and refold thermally denatured proteins following heat shock and play important roles in thermotolerance. The primary sequences of fungal Hsp104's contain a largely acidic C-terminal extension not present in bacterial ClpB's. In this work, deletion mutants were used to determine the role this extension plays in Hsp104 structure and function. Elimination of the C-terminal tetrapeptide DDLD diminishes binding of the tetratricopeptide repeat domain cochaperone Cpr7 but is dispensable for Hsp104-mediated thermotolerance. The acidic region of the extension is also dispensable for thermotolerance and for the stimulation of Hsp104 ATPase activity by poly-l-lysine, but its truncation results in an oligomerization defect and reduced ATPase activity in vitro. Finally, sequence alignments reveal that the C-terminal extension contains a sequence (VLPNH) that is conserved in fungal Hsp104's but not in other orthologues. Hsp104 lacking the entire C-terminal extension including the VLPNH region does not assemble and has very low ATPase activity. In the presence of a molecular crowding agent the ATPase activities of mutants with longer truncations are partially restored possibly through enhanced oligomer formation. However, elimination of the whole C-terminal extension results in an Hsp104 molecule which is unable to assemble and becomes aggregation prone at high temperature, highlighting a novel structural role for this region.
Mackay
Ryder G
RG
Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
Helsen
Christopher W
CW
Tkach
Johnny M
JM
Glover
John R
JR
D000222:Adaptation, Physiological; D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry*; D017931:DNA Primers; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006358:Hot Temperature; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D001704:Biopolymers; D017931:DNA Primers; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; C070972:HsP104 protein, S cerevisiae