The evolutionary conserved TLDc domain defines a new class of (H⁺)V-ATPase interacting proteins

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the vacuolar H+-ATPase (V-ATPase).
• Ncoa7 and Oxr1 possess a conserved TLDc domain and are implicated in oxidative stress response.
• The TLDc protein family includes Tbc1d24, Tldc1, and Tldc2, whose V-ATPase interactions were previously unknown.

Purpose of the Study:

• To investigate if Tbc1d24, Tldc1, and Tldc2 also interact with the V-ATPase.
• To determine if the TLDc domains are involved in these interactions.
• To identify the role of the TLDc motif in V-ATPase regulation.

Main Methods:

• Co-immunoprecipitation assays using endogenous and overexpressed proteins.
• GST pull-down assays with purified TLDc domains.
• Site-directed mutagenesis of conserved residues within the Ncoa7 TLDc domain.

Main Results:

• All five TLDc family members (Ncoa7, Oxr1, Tbc1d24, Tldc1, Tldc2) were found to interact with the V-ATPase.
• Purified TLDc domains of Ncoa7, Oxr1, and Tldc2 directly interacted with the V-ATPase.
• Specific point mutations in the Ncoa7 TLDc domain abolished or reduced V-ATPase interaction, highlighting the domain's importance.

Conclusions:

• The TLDc motif is a conserved interaction domain for the V-ATPase.
• All five members of the TLDc protein family interact with the V-ATPase.
• The TLDc motif defines a novel class of V-ATPase-interacting regulatory proteins.