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. 2000 Feb 15;19(4):741-8.
doi: 10.1093/emboj/19.4.741.

Hsp15: a ribosome-associated heat shock protein

P Korber  1 , J M Stahl, K H Nierhaus, J C Bardwell
Affiliations

Hsp15: a ribosome-associated heat shock protein

P Korber et al. EMBO J. .

Abstract

We are analyzing highly conserved heat shock genes of unknown or unclear function with the aim of determining their cellular role. Hsp15 has previously been shown to be an abundant nucleic acid-binding protein whose synthesis is induced massively at the RNA level upon temperature upshift. We have now identified that the in vivo target of Hsp15 action is the free 50S ribosomal subunit. Hsp15 binds with very high affinity (K(D) <5 nM) to this subunit, but only when 50S is free, not when it is part of the 70S ribosome. In addition, the binding of Hsp15 appears to correlate with a specific state of the mature, free 50S subunit, which contains bound nascent chain. This provides the first evidence for a so far unrecognized abortive event in translation. Hsp15 is suggested to be involved in the recycling of free 50S subunits that still carry a nascent chain. This gives Hsp15 a very different functional role from all other heat shock proteins and points to a new aspect of translation.

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Figures

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Fig. 1. Hsp15 associates with the free 50S subunit of ribosomes. Polysome profile of wild-type cells that were treated with 100 μg/ml chloramphenicol. A total of 17.5 A260 units of the lysate were loaded onto a sucrose gradient and fractionated as described in Materials and methods. The fractions indicated were analyzed by Western blotting. Hsp15 is present only in the fractions of the 50S subunit peak.
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Fig. 2. The influence of salt conditions on the binding of Hsp15 to 50S subunits and estimation of the dissociation constant. (A) Varied MgCl2 concentration at constant 150 mM NH4Cl. (B) Varied NH4Cl concentration at constant 10 mM MgCl2. See Figure 1 for the 10 mM MgCl2, 150 mM NH4Cl condition. The Western blot analyses of top, 30S and 50S peak fractions of the corresponding profiles are shown. Hsp15 is stably bound to the 50S subunit in and beyond the range of physiological ionic strength. (C) For the estimation of the dissociation constant (at 10 mM MgCl2, 150 mM NH4Cl), purified Hsp15 was added at 0.1 nM to various amounts of a chloramphenicol-treated lysate of Hsp15-depleted cells. For calibration, the total amount of Hsp15 in the binding reaction was loaded in parallel on the gel (‘100%’). ‘Int.’ is the fraction between the 30S and 50S subunit peaks. The exogenously added Hsp15 is again found in the 50S peak. A representative example is shown with Hsp15 and 50S concentrations of 0.1 and 2.0 nM, respectively. From five such reconstitution reactions, the KD was estimated to be in the nanomolar range, <5 nM.
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Fig. 3. (A) Polysome profile of wild-type cells that were not treated with chloramphenicol. Hsp15 is no longer bound to the free 50S subunit but is found in the top fractions. The gradient has been centrifuged for a longer time than in Figure 1 (see text). A total of 17.5 A260 units of the lysate were loaded onto the gradient. (B) The same lysate as in (A) was pre-incubated under dissociation conditions (1 mM MgCl2, 150 mM salt). 50S subunits that were generated by the dissociation bind Hsp15. A total of 5 A260 units of the lysate were loaded onto the gradient.
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Fig. 4. Only polysomes contain 50S subunits with high affinity for Hsp15. The fractions of run-off ribosomes and of polysomes from a wild-type lysate without chloramphenicol treatment (17.5 A260 units) were subjected to dissociation conditions (1 mM MgCl2, 150 mM salt). The same amounts (24 pmol) of 50S subunits thus generated were tested for their ability to bind purified Hsp15.
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Fig. 5. The reaction with puromycin abolishes the high affinity of 50S subunits for Hsp15. The same experiment as in Figure 3B was conducted additionally in the presence of puromycin (1 mM) and a combination of puromycin and chloramphenicol (1 and 0.3 mM, respectively). Chloramphenicol inhibits the puromycin reaction and preserves the 50S subunits with high affinity for Hsp15.
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Fig. 6. The affinity for Hsp15 of the 50S subunit can be generated by in vitro translation. In vitro translation of poly(U) was performed with 20 pmol of tight coupled 70S ribosomes. The ribosomes were dissociated after the translation reaction and incubated with Hsp15. The subunits thus generated were analyzed on sucrose gradients for the presence of radioactively labeled poly(phenylalanine) and Hsp15. (•), Complete translation reaction; (○), negative control, i.e. translation reaction without addition of energy. The UV absorption profile of the gradients is shown as a dashed line overlay on the radioactivity profile. Only after positive translation is a nascent chain generated that co-migrates with the 50S peak, and only in this case is Hsp15 found associated with the 50S subunit.
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Fig. 7. The high affinity Hsp15-binding state of the 50S subunit is induced under heat shock conditions. Lysates without chloramphenicol treatment were prepared from cells before and during heat shock (30→41°C) and the same amount (17.5 A260 units) was loaded onto the gradients. While only a part of the Hsp15 pool is found in the 50S peak before heat shock (for the extreme example of no binding at all compare Figure 3A), the majority of Hsp15 is bound to 50S during heat shock conditions.
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Fig. 8. A model explaining the nature of the 50S subunits that have high affinity for Hsp15. Hsp15 recognizes an off-pathway 50S subunit that still carries the nascent chain. It is the product of an erroneous dissociation of elongating ribosomes and has to be repaired for re-use in the translational cycle. For details see text.
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