Domain structure and lipid interaction of recombinant yeast Tim44 - PubMed (original) (raw)

Domain structure and lipid interaction of recombinant yeast Tim44

C Weiss et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE. To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied the properties of the pure protein and its interaction with model lipid membranes. Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain. The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone. Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region.

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Figures

Figure 1

Figure 1

Purification of recombinant yeast Tim44**_._** (Left) Purification on Ni-NTA-agarose. Lane 1, a lysate of bacteria overexpressing Tim44; lane 2, proteins not bound to Ni-NTA-agarose; lane 3, proteins eluted from the Ni-NTA-agarose with imidazole; lane 4, proteins remaining on the beads after imidazole elution. (Center) Anion-exchange chromatography on a MonoQ column. Fractions enriched in Tim44 (5 μl) were analyzed by SDS/PAGE. (Right) Pure Tim44 was loaded in lanes 1–3 (5, 10, or 20 μg, respectively). Proteins were stained with Coomassie blue. Mass, molecular mass.

Figure 2

Figure 2

Proteolysis of pure Tim44. Tim44 was digested with either proteinase K or trypsin for 2 min, and the products of proteolysis were separated by SDS/14% PAGE. The amount of proteinase K and trypsin added to each 50-μl reaction mixture is indicated at the top. The arrow indicates the position of full-length Tim44. The molecular mass (in kDa) of the standards is indicated on the right.

Figure 3

Figure 3

Binding of Tim44 to phospholipid vesicles. Pure Tim44 was incubated with vesicles composed of either CL and PC (1:5, wt/wt) or PC alone. The vesicles were collected by centrifugation, and protein associated with vesicles was detected by SDS/14% PAGE and staining with Coomassie blue. Bound Tim44 was quantified by densitometric integration of the peaks. Background “binding” (≈5% of Tim44 added without lipids) was subtracted, and the highest amount of vesicle-bound Tim44 (≈90% of Tim44 added) was taken as 100%. The continuous line represents the best fit of the data, assuming a dissociation constant of 129 μM and 13 mM for binding to PC/CL vesicles ● or PC vesicles ○, respectively.

Figure 4

Figure 4

Trypsinolysis of vesicle-bound Tim44. Pure Tim44 was incubated with no vesicles (Left), PC vesicles (Center), or CL/PC vesicles (Right). The indicated concentrations of trypsin were added, and the proteolysis products were analyzed by SDS/PAGE and staining with Coomassie blue.

Figure 5

Figure 5

Interaction of Tim44 with CL or PC monolayers. The initial surface pressure of both monolayers was adjusted to ≈20 mN/m. Pure Tim44 (0.8 mg/ml, 10 μl) was injected into the subphase, and the change in surface pressure was monitored as a function of time. (Dashed line) Tim44 was pretreated for 20 min with 6 μg of proteinase K before being injected into the subphase.

Figure 6

Figure 6

Tim44-induced surface-pressure increase as a function of initial surface pressure. The initial surface pressure of monolayers composed of CL (♦, ⋄, ▵, and □) or PC (▴) was set to the indicated values, and the change in surface pressure after Tim44 injection was recorded. Surface-pressure increase = (final surface pressure) − (initial surface pressure). Experiments were carried out at 100 mM (♦, ▴), 350 mM (⋄), 500 mM (▵), and 1 M NaCl (□).

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