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The intracellular pathway and assembly of newly formed variable surface glycoprotein of Trypanosoma brucei
Abstract:
Pulse-chase experiments using L-[35S]methionine suggest that Trypanosoma brucei MITat 1.2 variable surface glycoprotein (VSG) synthesized in the rough endoplasmic reticulum, a process that takes 6-8 min, is shuttled to the Golgi complex 8 min later. Labeling of ultrathin frozen sections with affinity-purified anti-cross-reacting determinant (CRD) IgG followed by protein A-colloidal gold shows that the CRD is localized in the trans-Golgi region. cis-Golgi is not labeled. VSG, when solubilized by treatment with the detergent Nonidet P-40, behaves on sucrose density gradients as a non-membrane protein with a sedimentation value of 5 S. In contrast, VSG solubilized in the presence of Zwittergent TM 3-14 yielded several VSG-containing fractions greater than 5 S, and only the 5S fraction contained the CRD. Lack of the CRD in VSG complexes with sedimentation values greater than 5 S suggests that this determinant is either masked from antibody, perhaps by involvement in polymer formation, or represents the membrane form of VSG recently described by Cardoso de Almeida and Turner [Cardoso de Almeida, M. L. & Turner, M. J. (1983) Nature (London) 302, 349-352].
Insights
Trypanosoma brucei variable surface glycoprotein (VSG) moves from the endoplasmic reticulum to the Golgi. The cross-reacting determinant (CRD) is found in the trans-Golgi, suggesting it may be masked in VSG complexes.
Area of Science:
- Cell Biology
- Parasitology
- Molecular Biology
Background:
- Trypanosoma brucei undergoes antigenic variation mediated by surface glycoproteins.
- Understanding the trafficking and post-translational modification of VSG is crucial for parasitic evasion mechanisms.
Purpose of the Study:
- To investigate the intracellular transport and localization of the MITat 1.2 variable surface glycoprotein (VSG) in Trypanosoma brucei.
- To determine the location of the cross-reacting determinant (CRD) during VSG processing.
Main Methods:
- Pulse-chase experiments with L-[35S]methionine to track VSG synthesis and transport.
- Immunolabeling of ultrathin frozen sections using anti-CRD IgG and protein A-colloidal gold.
- Sucrose density gradient centrifugation of VSG solubilized with different detergents.
Main Results:
- VSG is synthesized in the rough endoplasmic reticulum and transported to the Golgi complex within 14-16 minutes.
- The CRD is localized specifically to the trans-Golgi region, with no labeling in the cis-Golgi.
- Detergent solubilization revealed that CRD-containing VSG exists as a 5S non-membrane form, while other fractions (>5S) lacked CRD.
Conclusions:
- The CRD is associated with the soluble form of VSG in the trans-Golgi.
- The absence of CRD in larger VSG complexes suggests it may be masked or represent a membrane-associated form.