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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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Lck membrane-binding kinetics in the presence and absence of CD4. (A) CD4-negative Jurkat cells were labeled for 5 min  and chased as indicated. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions as described for Fig. 2 B. ★ indicates the background band. (B) The same experiments as in A, now for CD4-positive Jurkat cells. (C) Cell lysates of CD4-negative (−)  and CD4-positive (+) Jurkat cells were analyzed for CD4 expression by immunoblotting. (D) Quantitation of Lck membrane-binding  experiments for two CD4-negative and one CD4-positive Jurkat cell line. Autoradiograms were digitized (as described in Materials and  Methods) and analyzed using the Molecular Analyst program (Bio-Rad). Membrane-associated Lck is expressed as a percentage of the  total amount of Lck and plotted against time.
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Figure 3: Lck membrane-binding kinetics in the presence and absence of CD4. (A) CD4-negative Jurkat cells were labeled for 5 min and chased as indicated. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions as described for Fig. 2 B. ★ indicates the background band. (B) The same experiments as in A, now for CD4-positive Jurkat cells. (C) Cell lysates of CD4-negative (−) and CD4-positive (+) Jurkat cells were analyzed for CD4 expression by immunoblotting. (D) Quantitation of Lck membrane-binding experiments for two CD4-negative and one CD4-positive Jurkat cell line. Autoradiograms were digitized (as described in Materials and Methods) and analyzed using the Molecular Analyst program (Bio-Rad). Membrane-associated Lck is expressed as a percentage of the total amount of Lck and plotted against time.

Mentions: Therefore, we also determined Lck membrane-binding kinetics in the CD8-negative Jurkat cells. Three different Jurkat clones were screened by FACS® analysis for CD4 expression. Two were found to express very little if any CD4, whereas one expressed considerable amounts of CD4 (approximately half the amount of CD4 in SupT1 cells, which is ∼30,000 copies per cell; Pelchen-Matthews et al., 1991). This was confirmed by immunoblotting (Fig. 3 C). Again, we did not find a difference in membrane association of newly synthesized Lck between CD4-positive and -negative cells (Fig. 3, A, B, and D). However, membrane-binding kinetics of Lck were slower in Jurkat cells than in SupT1 cells (t1/2 21 min in Jurkat vs. 9 min in SupT1), indicating that cell type–specific differences can influence these kinetics.


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)

Lck membrane-binding kinetics in the presence and absence of CD4. (A) CD4-negative Jurkat cells were labeled for 5 min  and chased as indicated. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions as described for Fig. 2 B. ★ indicates the background band. (B) The same experiments as in A, now for CD4-positive Jurkat cells. (C) Cell lysates of CD4-negative (−)  and CD4-positive (+) Jurkat cells were analyzed for CD4 expression by immunoblotting. (D) Quantitation of Lck membrane-binding  experiments for two CD4-negative and one CD4-positive Jurkat cell line. Autoradiograms were digitized (as described in Materials and  Methods) and analyzed using the Molecular Analyst program (Bio-Rad). Membrane-associated Lck is expressed as a percentage of the  total amount of Lck and plotted against time.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2185081&req=5

Figure 3: Lck membrane-binding kinetics in the presence and absence of CD4. (A) CD4-negative Jurkat cells were labeled for 5 min and chased as indicated. Lck was immunoprecipitated from membrane (M) and soluble (S) fractions as described for Fig. 2 B. ★ indicates the background band. (B) The same experiments as in A, now for CD4-positive Jurkat cells. (C) Cell lysates of CD4-negative (−) and CD4-positive (+) Jurkat cells were analyzed for CD4 expression by immunoblotting. (D) Quantitation of Lck membrane-binding experiments for two CD4-negative and one CD4-positive Jurkat cell line. Autoradiograms were digitized (as described in Materials and Methods) and analyzed using the Molecular Analyst program (Bio-Rad). Membrane-associated Lck is expressed as a percentage of the total amount of Lck and plotted against time.
Mentions: Therefore, we also determined Lck membrane-binding kinetics in the CD8-negative Jurkat cells. Three different Jurkat clones were screened by FACS® analysis for CD4 expression. Two were found to express very little if any CD4, whereas one expressed considerable amounts of CD4 (approximately half the amount of CD4 in SupT1 cells, which is ∼30,000 copies per cell; Pelchen-Matthews et al., 1991). This was confirmed by immunoblotting (Fig. 3 C). Again, we did not find a difference in membrane association of newly synthesized Lck between CD4-positive and -negative cells (Fig. 3, A, B, and D). However, membrane-binding kinetics of Lck were slower in Jurkat cells than in SupT1 cells (t1/2 21 min in Jurkat vs. 9 min in SupT1), indicating that cell type–specific differences can influence these kinetics.

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