ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Trafficking

Background:

• Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) are crucial for post-Golgi traffic.
• While human and yeast GGAs share functional domains, their sequence identity is low (~25%).
• The ARF-binding region is the most conserved domain across GGA proteins.

Purpose of the Study:

• To investigate the role of ARF interaction in GGA protein localization and function.
• To compare the necessity of ARF binding for human and yeast GGA proteins.
• To identify key domains responsible for yeast GGA localization and function.

Main Methods:

• Site-directed mutagenesis to create non-ARF-binding GGA mutants in yeast and mammalian cells.
• Assays for carboxypeptidase Y missorting and synthetic lethality in yeast.
• Localization studies using green fluorescent protein (GFP)-tagged yeast Gga1p and truncation analysis.
• Observation of mammalian GGA localization in cells expressing mutated proteins.

Main Results:

• A point mutation disrupting ARF binding impaired human GGA localization but not yeast Gga function.
• Yeast Gga mutants lacking ARF interaction showed normal carboxypeptidase Y sorting and viability.
• ARF interaction was not essential for yeast Gga1p localization to the Golgi.
• N-terminal VHS and C-terminal hinge/ear domains were critical for yeast Gga localization and function.

Conclusions:

• Yeast and mammalian GGAs exhibit differential requirements for ARF interaction in Golgi localization and function.
• Yeast GGAs can assemble functional protein complexes at the late Golgi independently of ARF binding.
• Mammalian GGAs require ARF interaction for proper Golgi localization and function.
• This study reveals distinct evolutionary strategies for ARF-GGA interaction in membrane trafficking.