citations

                    Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes.
                

Authors:

Zoltner M, Leung KF, Alsford S, Horn D, Field MC

In:

Source: PLoS Pathog
Publication Date: (2015)
Issue: 11(10): e1005236

Research Area:

Parasitology

Experiment

3x10e7 bloodstream-form cells were harvested by centrifugation at 800 x g for 10 min at 4°C. Cells were resuspended in 100 ul of Amaxa Human T-cell Nucleofector solution (VPA-1002) at 4°C, mixed with 10 ug (in 5 ul) of linearised plasmid DNA and transferred to electrocuvettes. Transfection was achieved using an Amaxa Nucleofector II with Program X-001. Cells were then transferred to Tube A containing 30 ml of HMI-9 medium plus any appropriate antibiotic drug for parental cell growth. Serial dilution was performed by transferring 3 ml of cell suspension from Tube A into Tube B containing 27 ml of HMI-9 medium and repeated again by diluting 3 ml from Tube B into Tube C. 1 ml aliquots for each dilution were distributed between three 24-well plates and incubated at 37°C. After 6 h, HMI-9 containing antibiotic selection was added to the wells at the desired final concentration. Transformed cells were recovered on day 5–6 post-transfection.

Abstract

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.

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