Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

Abstract

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Fig. 1.

Biochemical characterization of PFO*. (A) SDS/PAGE of purified recombinant PFO*. Protein was purified as described in Materials and Methods. An aliquot (1 μg) was subjected to 8% SDS/PAGE, and proteins were visualized with Coomassie Brilliant blue R-250. (B) Lysis of SV-589 cells by PFO, but not by PFO*, as measured by release of LDH. On day 0, cells were set up in 12-well plates as described in SI Materials and Methods. On day 3, each monolayer received 0.5 mL PBS containing varying amounts of the indicated protein. After incubation for 1 h at 4 °C, the PBS was removed and assayed for LDH as described in Materials and Methods. (C) Immunoblot analysis of two cytosolic proteins released from SV-589 cells after incubation with varying amounts of purified PFO, but not PFO*. Aliquots of the cell-free PBS solution from B (lanes 1–7) and from the total cell lysate (lane 8) were immunoblotted with antibodies directed against the ubiquitin activating enzyme E1 and LDH. Before immunoblot analysis, the total cell lysate was adjusted to the same volume as each cell-free PBS solution. Blots were exposed to film at room temperature for 30–60 s.

Fig. 2.

Binding of ¹²⁵I-PFO* to SV-589 cells. On day 0, cells were set up in medium A at 1 × 10⁵ cells per 60-mm dish. On day 3, cells were refed with medium A. On day 4, cells were treated with fresh medium B with or without 2% (wt/vol) HPCD for 1 h at 37 °C, after which the cells were washed five times as described in Materials and Methods and then incubated at 4 °C for the indicated time with 2 mL of ice-cold buffer E containing either 100 μg/mL ¹²⁵I-PFO* (6.5 × 10³ cpm/μg protein) (A) or for 2 h with the indicated concentration of ¹²⁵I-PFO* (10 × 10³cpm/μg) (B). The total amount of ¹²⁵I- PFO* bound to the cells was determined as described in Materials and Methods. Each value represents the average of duplicate incubations. The values (mean ± SEM) for total cellular protein content did not differ significantly in cells treated with or without HPCD (0.59 ± 0.02 and 0.56 ± 0.02 mg per dish, respectively).

Fig. 3.

¹²⁵I-PFO* binding to SV-589 cells after prior treatment with different sterols. On day 0, cells were set up in medium A at 1 × 10⁵ cells per 60-mm dish. On day 2, cells were switched to lipoprotein-deficient medium C. On day 3, cells were refed with medium C containing 10 μM compactin and 50 μM sodium mevalonate and then incubated for 16 h at 37 °C. On day 4, each dish received fresh medium C containing 10 μM compactin, 50 μM sodium mevalonate, and varying concentrations of the indicated sterol complexed with MCD (sterol/MCD molar ratio of 1:12). After incubation for 3 h at 37 °C, the cells were washed five times as described in Materials and Methods and then incubated with 2 mL ice-cold buffer E containing 5 μg/mL ¹²⁵I-PFO* (30 × 10³ cpm/μg) for 30 min at 4 °C. The total amount of cell surface ¹²⁵I-PFO* binding was determined as described in Materials and Methods. Each value represents the average of duplicate incubations.

Fig. 4.

Movement of LDL-derived cholesterol from lysosomes to plasma membrane as determined by cell surface ¹²⁵I-PFO* binding. On day 0, SV-589 cells were set up in medium A at 1 × 10⁵ cells per 60-mm dish. On day 2, cells were switched to lipoprotein-deficient medium C. On day 3, cells were refed with medium C containing 50 μM compactin and 50 μM sodium mevalonate and incubated for 16 h at 37 °C. On day 4, cells received fresh medium E containing 50 μM compactin, 50 μM mevalonate, and one of the following treatments in the absence or presence of 100 μM chloroquine: (A) 50 μg protein/mL LDL for the indicated time; (B) the indicated concentration of LDL for 5 h; or (C) the indicated concentration of cholesterol/MCD complex. After incubation for 5 h at 37 °C, the cells were washed five times as described in Materials and Methods and then incubated with 2 mL of ice-cold buffer E containing 25 μg/mL ¹²⁵I-PFO* (6 × 10³ cpm/μg). After 2 h at 4 °C, the total amount of cell surface binding of ¹²⁵I-PFO* was determined. Each value represents the average of duplicate incubations.

Fig. 5.

Reduced delivery of LDL-derived cholesterol to the cell surface of fibroblasts from subjects with NPC1 disease, Wolman disease, and homozygous FH. On day 0, diploid fibroblasts from the indicated subject were set up in medium A at 2 × 10⁴ (control), 4 × 10⁴ (NPC1 and Wolman), and 8 × 10⁴ (FH) cells per 60-mm dish, respectively. On day 3, cells were refed with the same medium. On day 5, cells were switched to lipoprotein-deficient medium D. On day 6, cells were refed with medium D containing 50 μM compactin and 50 μM sodium mevalonate. On day 7, each dish was refed with medium F containing 50 μM compactin, 50 μM mevalonate, and the indicated amount of LDL (A) or 50 μM cholesterol/MCD complex (B). After 5 h at 37 °C, each monolayer was washed 5 times as described in Materials and Methods and then incubated with 2 mL ice-cold buffer E containing 5 μg/mL ¹²⁵I-PFO* (23 × 10³ cpm/μg). After 30 min at 4 °C, the total amount of cell surface ¹²⁵I-PFO* binding was determined. (A and B) Each value represents the average of duplicate incubations.

Fig. 6.

Relation between cholesterol content of plasma membrane and amount of ¹²⁵I-PFO* bound to cell surface of SV-589 cells. (A) On day 0, cells were set up in medium A at 4 × 10⁵ cells per 100-mm dish. On day 3, cells were refed with medium B. On day 4, plasma membranes were purified as described in Materials and Methods. Equal volumes of the input, unbound fraction, and bound (eluted) fraction were subjected to 8 or 15% SDS/PAGE and immunoblotted with antibodies against the indicated organelle marker. Filters were exposed to film at room temperature for 5–60 s. (B) On day 0, cells were set up in medium A at 1 × 10⁵ cells per 60-mm dish. On day 3, cells were refed with medium B. On day 4, groups of 12 dishes of cells were each treated with the indicated concentration of HPCD in medium B for 1 h at 37 °C, after which the cells were washed five times as described in Materials and Methods. Each group of 12 dishes was then divided into two sets of six dishes. The cells from each six-dish set were pooled together and processed for purification of plasma membranes. Lipids were extracted from the plasma membranes, and the content of unesterified cholesterol and choline-containing phospholipids was measured. Values for total phospholipids were calculated from measurements of choline-containing phospholipids as described in Materials and Methods. Each value represents the average of duplicate measurements for each of the two pooled sets, each denoted by a different symbol (●, △). (C) Cells were cultured under identical condition as described in B. On day 4, after treatment with HPCD and washing, each cell monolayer was incubated with 2 mL of ice-cold buffer E containing 25 μg/mL ¹²⁵I-PFO* (19 × 10³ cpm/μg). After 2 h at 4 °C, the total amount of cell surface ¹²⁵I-PFO* binding was determined. Each value represents the average of duplicate incubations. (D) Graph showing the amount of cell surface ¹²⁵I-PFO* binding plotted as a function of the unesterified cholesterol content of the plasma membrane for three independent experiments (denoted by red, blue, and black circles), one of which (●) was done in parallel with B and C.