Functions of the Yeast GTPase-Activating Proteins Age1 and Gcs1 for Post-Golgi Vesicular Transport
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Organelles within the endomembrane system of all eukaryotic cells exchange membrane lipids and proteins using membrane-bound transport vesicles. This highly conserved vesicular transport process is essential for life and is highly regulated. Much of this regulation is provided by small monomeric GTP-binding proteins such as Arf and Arl that act as molecular switches, cycling between inactive GDP-bound and active GTP-bound states. This cycle of GTP binding and hydrolysis is controlled by guanine-nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. I have investigated regulatory interactions involving two ArfGAPs, Age1 and Gcs1, involved in post-Golgi vesicular transport in the budding yeast Saccharomyces cerevisiae. In yeast, the Age2 + Gcs1 ArfGAP pair is essential and facilitates post-Golgi transport. I found that overexpression of either the poorly characterized ArfGAP Age1 or the Sfh2 phosphatidylinositol-transfer protein can bypass the requirement for Age2 and Gcs1. Indeed, endogenous Age1 is required for efficient Sfh2-bypass. Moreover, the yeast phospholipase D protein, Spo14, which is activated by Sfh2 and regulates membrane lipid composition, is required for Age1 to effectively alleviate the deleterious effects of defective Age2 + Gcs1 function. My findings suggest that Age1 is regulated by membrane lipid composition and can provide ArfGAP function for post-Golgi transport. Gcs1 is involved in multiple vesicular transport stages, is a dual-specificity GAP for both Arf and Arl1 proteins and, as shown here, also has functions independent of its GAP activity. The absence of Gcs1 causes cold sensitivity for growth and endocytic transport. The cold sensitivity of cells lacking Gcs1 is alleviated by the elimination of either the Arl1 or Ypt6 vesicle-tethering pathway at the trans-Golgi, or by overexpression of Imh1, an effector of the Arl1 pathway. I found elimination of the Ypt6 pathway also prevents Arl1 activation and membrane localization, that Arl1 binding by Imh1 is necessary and sufficient for alleviation, and that the Gcs1 function required for growth and transport in the cold is independent of any GAP activity. My findings suggest that in the absence of this GAP-independent function of Gcs1 the resulting dysregulated Arl1 causes the gcs1? defects through the sequestration of a yet-to-be-determined cellular factor.