The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Trafficking

Background:

• Ypt1 GTPase regulates ER-to-Golgi transport, while Ypt31/32 GTPases are vital for trans-Golgi exit.
• A previously identified Ypt1 guanine nucleotide exchange factor (GEF) activity is a large, salt-extractable, membrane-associated complex.
• The TRAPP complex is implicated in ER-to-Golgi transport and shares properties with the Ypt1 GEF.

Purpose of the Study:

• To investigate whether the TRAPP complex possesses Ypt1 GEF activity.
• To determine if TRAPP influences other Ypt GTPases involved in Golgi transport.
• To explore the potential role of TRAPP as a common GEF coordinating Golgi entry and exit.

Main Methods:

• Yeast cell culture and expression of GST-tagged TRAPP subunits (Bet3p, Bet5p).
• Glutathione-agarose precipitation to isolate TRAPP-associated GEF activity.
• In vitro assays measuring GDP release and GTP uptake by Ypt1p and Ypt31/32 GTPases.
• Inhibition assays using a dominant-negative Ypt1 mutant (Ypt1-D124N).
• Coprecipitation and overexpression studies to assess in vivo function.

Main Results:

• TRAPP exhibits significant Ypt1 GEF activity, with the majority of activity associated with the TRAPP complex (>670 kDa).
• TRAPP also stimulates nucleotide exchange on Ypt31/32 GTPases but not on Sec4p.
• The Ypt1-D124N mutant inhibits both Ypt1 and Ypt32 GEF activities of TRAPP.
• In vivo studies suggest TRAPP functions as a GEF for both Ypt1 and Ypt31/32.

Conclusions:

• TRAPP possesses Ypt1 GEF activity, confirming its role in early Golgi transport.
• TRAPP also acts as a GEF for Ypt31/32, suggesting a broader role in Golgi dynamics.
• A common GEF complex (TRAPP) for Ypt1 and Ypt31/32 may coordinate protein transport into and out of the Golgi.