Lhs1p - an assistance partner for Kar2p in translocation and
protein refolding in the endoplasmic reticulum.
Anton Shmelev,
and Marja Makarow
Institute of Biotechnology, University of
Helsinki, PL56, Helsinki, HY-00014, Finland
(anton.shmelev@helsinki.fi, marja.makarow@helsinki.fi)
Lhs1p is involved in protein translocation and refolding of
heat-denatured glycoproteins in ER, and in acquisition of
thermotolerance. Point mutations were made using structural model of
putative ATPase domain (ATPdom) to study domain role in Lhs1p
functions. Multicopy but not single copy expression of the mutants or
ATPdom alone rescued translocation of reporter protein, retarded in
lhs1 deletant. Fully functional in translocation, refolding and
thermotolerance acquisition, His6-tagged Lhs1p had no
detectable ATPase activity when purified from microsomes. Unlike
Kar2p, Lhs1p was not activated by GST-fusions of the J-domains of ER
proteins Sec63p, Scj1p, and Jem1p, or the nucleotide exchange factor
Sil1p, as they did not bind to Lhs1p even with a 15 aa synthetic
peptide or ATP present. The Lhs1p ATPdom could not functionally
replace that of Kar2p. When fused to the peptide binding domain (PBD)
of Kar2p, it did not suppress ts lethality of kar2
mutants, and not substitute Lhs1p in ER translocation. DSP
crosslinking indicated that Lhs1p and its mutants, but not ER
localised ATPdom alone, interacted with Kar2p. C-terminal fragment of
the Lhs1p PBD (aa 836-881) fused to ATPdom restored binding to Kar2p,
but not translocation defect. Thus, Lhs1p does not share the
activities of Hsp70s. Its ATPdom participates in translocation
directly, but PBD indirectly via collaboration with Kar2p. Lh1p might
assist Kar2p in ER translocation and/or Ire1p derepression in Hsp90
manner.