Biochemical methods for studying kinetic regulation of Arf1 activation by Sec7.

Abstract

The ADP ribosylation factor (Arf) family of small guanosine triphosphatases (GTPases) regulates vesicular transport at several locations within the cell, and is in turn regulated by guanine nucleotide exchange factors (GEFs) via a conserved catalytic domain, termed the Sec7 domain. The catalytic activity of the Sec7 domain is well characterized in the context of a few GEFs acting at the periphery of the cell. This chapter describes the techniques used to extend the biochemical analysis of activity to the much larger GEFs acting on the Arf family in the core secretory pathway, using the activity of Saccharomyces cerevisiae Sec7 on Arf1, regulating export from the trans-Golgi network, as a model. The complete methods for purification to near homogeneity of all proteins required, including several Sec7 constructs and multiple relevant small GTPases, are detailed. These are followed by methods for the quantification of the nucleotide exchange activity of Sec7 in a physiologically relevant context, including modifications required to dissect the signal integration functions of Sec7 as an effector of several other small GTPases, and methods for identifying stable Sec7-small GTPase interactions in the presence of membranes. These techniques may be extended to the analysis of similar members of the Sec7 GEF subfamily in other species and acting elsewhere in the secretory pathway.