PfHsp70-1 is stable and soluble when expressed in <i>S. cerevisiae</i>.

<p>(A) A cycloheximide chase analysis was performed using a wild type yeast strain transformed with plasmid p416-P<i>_SSA</i>₁-<i>PfHsp70-1</i> (top) or p416-P<i>_SSA</i>₁- <i>SSA1</i> (bottom). The assay was performed at 30°C, and samples were taken at the indicated time points after the addition of cycloheximide. Samples were processed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#s2" target="_blank">Methods</a>”. (B) Cells expressing PfHsp70-1 were lysed and processed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#s2" target="_blank">Methods</a>”. Sec61 was probed as an ER membrane protein control and G6PDH was probed as a soluble (cytosolic) protein control. L: lysate; S, soluble fraction; P, pellet fraction. Please note that the lysate examined in this panel represents only a variable fraction of the total lysate that was processed to obtain the soluble and pellet samples.</p

Mutations in the ATPase domain and in the SBD subvert the PfHsp70-1-mediated improvement of growth in the <i>ssa1-45</i> or <i>ssa1Δssa2Δ</i> mutants.

<p>(A) The indicated mutant strains transformed with the designated plasmids were grown to log phase, serially diluted, and plated on selective media and incubated at the indicated temperatures for 2–3 d. (B) Cells were plated on selective media containing the indicated stress-inducing agent, grown at 26°C, and imaged after 3 d. In both panels, the images shown are representative of at least three independent experiments, all demonstrating the same phenomena. “-” indicates an empty vector control.</p

PfHsp70-1 partially restores translocation and ERAD efficiencies in the <i>ssa1-45</i> mutant.

<p>(A) The indicated transformed strains were grown to log phase at 26°C, and were then incubated at 37°C for 15 min. Equal amounts of cells were harvested and processed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#s2" target="_blank">Methods</a>”. The graph shows the relative accumulation of ppαF. The accumulation of ppαF in the <i>ssa1-45</i> strain transformed with an empty vector was set to 1 (i.e., maximal accumulation of ppαF). Error bars indicate SEM, n = 4 independent experiments, and statistical significance was calculated using a Student's t-test; * denotes p<0.05, and ** denotes p<0.005. (B) ERAD was examined using cycloheximide chase analyses as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#s2" target="_blank">Methods</a>”. The percent CFTR remaining over time was calculated for <i>ssa1-45</i> transformed with an empty vector (○), an Ssa1 expression vector (•), or a PfHsp70-1 expression vector (▵). CFTR degradation in the wild type <i>SSA1</i> strain was identical to that of <i>ssa1-45</i> transformed with the Ssa1 expression vector (data not shown). Error bars depict the SEM, n≥4, and statistical significance was calculated using a Student's t-test; * denotes p<0.05.</p

Ssa1 is not upregulated in response to wild type or mutant PfHsp70-1 expression.

<p>The indicated yeast strains transformed with the indicated plasmids were grown in liquid culture to log phase at 26°C and were then incubated at 37°C for 30 min. Equivalent amounts of cells were harvested, and samples were processed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#s2" target="_blank">Methods</a>”. Note: The anti-Ssa1 antiserum crossreacts with Ssa3 and/or Ssa4, since there is a faint band in the <i>ssa1Δssa2Δ</i> strain transformed with the empty vector. This antiserum recognizes the last 56 amino acids in Ssa1 (E. Craig, personal communication). Also note that the levels of the PfHsp70-1 mutants are similar to the level of the wild type protein (compare the last three lanes, top-right panel). The absence of an increased level of Ssa1 in <i>ssa1-45</i> yeast containing the <i>SSA1</i> expression vector most likely results from the fact that the protein binds to its message and represses translation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#pone.0020047-Stone1" target="_blank">[71]</a>. Thus, the expression of Ssa1 is expected to depress both its own synthesis and the synthesis of the Ssa1-45 mutant protein.</p

Yeast Expression Plasmids used in this study.

<p>Yeast Expression Plasmids used in this study.</p

Yeast Strains used in this study.

<p>Yeast Strains used in this study.</p

<i>S. cerevisiae</i> Hsp70, Ssa1, and <i>P. falciparum</i> Hsp70, PfHsp70-1, are >70% identical.

<p>The alignment was generated using ClustalW2 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020047#pone.0020047-Thompson1" target="_blank">[70]</a>. Sequences in bold face indicate identity, and underlined sequences indicate similarity. Gaps are indicated with a dash. Asterisks denote amino acids substituted in the PfHsp70-1 mutants (see text for details).</p