Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol.
We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In DeltarpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5-10% and 20-30% of total protein aggregated at 30 degrees C and 42 degrees C respectively. The aggregates contained 350-400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30 degrees C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low-level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to DeltarpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30 degrees C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK-depleted cells. At 42 degrees C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42 degrees C.
Tomoyasu
T
T
Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
Mogk
A
A
Langen
H
H
Goloubinoff
P
P
Bukau
B
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D012808:Sigma Factor; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; C063842:dnaK protein, E coli