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Trafficking of an acylated cytosolic protein: newly synthesized p56(lck) travels to the plasma membrane via the exocytic pathway.

Bijlmakers MJ, Marsh M - J. Cell Biol. (1999)

Bottom Line: We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later.Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A.From this location Lck is transported to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom.

ABSTRACT
The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.

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Membrane-binding kinetics of Lck. (A) SupT1 cells  were broken in hypotonic buffer  and Dounce homogenized. After removal of nuclei (5 min at  1,500 rpm), membrane and soluble fractions were separated by  centrifugation at 100,000 g.  Equivalent amounts of total (T),  nuclear (N), membrane (M),  and soluble (S) fractions were  analyzed by immunoblotting  with LckC. (B) SupT1 cells (2 ×  108) were labeled with [35S]methionine/cysteine (2 mCi) for 5  min and chased for the indicated  times. Lck was immunoprecipitated from the membrane (M)  and soluble (S) fractions with  LckN and analyzed by SDS-PAGE and autoradiography.  Lck and CD4 are indicated. ★  indicates the background band.
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Figure 2: Membrane-binding kinetics of Lck. (A) SupT1 cells were broken in hypotonic buffer and Dounce homogenized. After removal of nuclei (5 min at 1,500 rpm), membrane and soluble fractions were separated by centrifugation at 100,000 g. Equivalent amounts of total (T), nuclear (N), membrane (M), and soluble (S) fractions were analyzed by immunoblotting with LckC. (B) SupT1 cells (2 × 108) were labeled with [35S]methionine/cysteine (2 mCi) for 5 min and chased for the indicated times. Lck was immunoprecipitated from the membrane (M) and soluble (S) fractions with LckN and analyzed by SDS-PAGE and autoradiography. Lck and CD4 are indicated. ★ indicates the background band.

Mentions: After pulse–chase labeling, cells were incubated in 1 ml hypotonic buffer (20 mM Tris, pH 7.8, 2 mM MgCl2, 1 mM EDTA, 1 mM PMSF, CLAP as above) on ice for 12 min and homogenized by 15 strokes in a Dounce homogenizer (Wheaton Scientific). To remove nuclei, cell homogenates were centrifuged for 5 min at 1,500 rpm, 4°C. The postnuclear supernatant was centrifuged in an Optima TL Ultracentrifuge (Beckman Instruments) for 45 min at 100,000 g, 4°C, to recover total cellular membranes. The pellet (membrane fraction) was resuspended in hypotonic buffer, Dounce homogenized (20 strokes), and adjusted to 2% NP-40, 150 mM NaCl, 1 mM PMSF, and CLAP. Similarly, the soluble fractions were adjusted to 2% NP-40 and 150 mM NaCl. The final volume of both fractions was equivalent. Samples (2% of total volume) were analyzed by immunoblotting to check the efficiency of membrane separation and the remainder was subjected to immunoprecipitation. In control experiments, to examine the presence of Lck in the nuclear fraction, nuclei were resuspended in an equal volume of the same buffer as the membrane and soluble fractions and analyzed for Lck by immunoprecipitation and immunoblotting. The amount of Lck detected in this fraction was negligible (Fig. 2 A).


Trafficking of an acylated cytosolic protein: newly synthesized p56(lck) travels to the plasma membrane via the exocytic pathway.

Bijlmakers MJ, Marsh M - J. Cell Biol. (1999)

Membrane-binding kinetics of Lck. (A) SupT1 cells  were broken in hypotonic buffer  and Dounce homogenized. After removal of nuclei (5 min at  1,500 rpm), membrane and soluble fractions were separated by  centrifugation at 100,000 g.  Equivalent amounts of total (T),  nuclear (N), membrane (M),  and soluble (S) fractions were  analyzed by immunoblotting  with LckC. (B) SupT1 cells (2 ×  108) were labeled with [35S]methionine/cysteine (2 mCi) for 5  min and chased for the indicated  times. Lck was immunoprecipitated from the membrane (M)  and soluble (S) fractions with  LckN and analyzed by SDS-PAGE and autoradiography.  Lck and CD4 are indicated. ★  indicates the background band.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2185081&req=5

Figure 2: Membrane-binding kinetics of Lck. (A) SupT1 cells were broken in hypotonic buffer and Dounce homogenized. After removal of nuclei (5 min at 1,500 rpm), membrane and soluble fractions were separated by centrifugation at 100,000 g. Equivalent amounts of total (T), nuclear (N), membrane (M), and soluble (S) fractions were analyzed by immunoblotting with LckC. (B) SupT1 cells (2 × 108) were labeled with [35S]methionine/cysteine (2 mCi) for 5 min and chased for the indicated times. Lck was immunoprecipitated from the membrane (M) and soluble (S) fractions with LckN and analyzed by SDS-PAGE and autoradiography. Lck and CD4 are indicated. ★ indicates the background band.
Mentions: After pulse–chase labeling, cells were incubated in 1 ml hypotonic buffer (20 mM Tris, pH 7.8, 2 mM MgCl2, 1 mM EDTA, 1 mM PMSF, CLAP as above) on ice for 12 min and homogenized by 15 strokes in a Dounce homogenizer (Wheaton Scientific). To remove nuclei, cell homogenates were centrifuged for 5 min at 1,500 rpm, 4°C. The postnuclear supernatant was centrifuged in an Optima TL Ultracentrifuge (Beckman Instruments) for 45 min at 100,000 g, 4°C, to recover total cellular membranes. The pellet (membrane fraction) was resuspended in hypotonic buffer, Dounce homogenized (20 strokes), and adjusted to 2% NP-40, 150 mM NaCl, 1 mM PMSF, and CLAP. Similarly, the soluble fractions were adjusted to 2% NP-40 and 150 mM NaCl. The final volume of both fractions was equivalent. Samples (2% of total volume) were analyzed by immunoblotting to check the efficiency of membrane separation and the remainder was subjected to immunoprecipitation. In control experiments, to examine the presence of Lck in the nuclear fraction, nuclei were resuspended in an equal volume of the same buffer as the membrane and soluble fractions and analyzed for Lck by immunoprecipitation and immunoblotting. The amount of Lck detected in this fraction was negligible (Fig. 2 A).

Bottom Line: We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later.Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A.From this location Lck is transported to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom.

ABSTRACT
The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.

Show MeSH
Related in: MedlinePlus