Systematic Structure-Function Analysis of the Small GTPase Arf1 in Yeast

Abstract

Members of the ADP-ribosylation factor (Arf) family of small GTPases are implicated in vesicle traffic in the secretory pathway, although their precise function remains unclear. We generated a series of 23 clustered charge-to-alanine mutations in the Arf1 protein ofSaccharomyces cerevisiae to determine the portions of this protein important for its function in cells. These mutants display a number of phenotypes, including conditional lethality at high or low temperature, defects in glycosylation of invertase, dominant lethality, fluoride sensitivity, and synthetic lethality with thearf2 null mutation. All mutations were mapped onto the available crystal structures for Arf1p: Arf1p bound to GDP, to GTP, and complexed with the regulatory proteins ArfGEF and ArfGAP. From this systematic structure-function analysis we demonstrate that all essential mutations studied map to one hemisphere of the protein and provide strong evidence in support of the proposed ArfGEF contact site on Arf1p but minimal evidence in support of the proposed ArfGAP-binding site. In addition, we describe the isolation of a spatially distant intragenic suppressor of a dominant lethal mutation in the guanine nucleotide-binding region of Arf1p.

Missense-depleted regions in population exomes implicate ras superfamily nucleotide-binding protein alteration in patients with brain malformation

“…In addition, the affected residue p.K127 is known to be important in functional studies of ARF1 in other systems. 21 , 22 A third patient had a de novo missense variant in ARF1 c.296G>A (p.R99H) and had brain malformation with delayed myelination in childhood and significant cerebral underdevelopment 12 years later, and p.R99 appears to contact two of the GDP-binding residues of ARF1 by LPC/CSU analysis ( Figure 3 and Supplementary Table 7 ). The three ARF1 missense variants were in MDR ( Supplementary Figure 5 ).…”

Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins

“…These findings sharply contrast with the effects of altering cognate P-loop–motif lysines in yeast Arf1p and the rab Ypt1p, which yielded severe phenotypes equivalent to complete gene inactivation (Wagner et al. , 1987; Click et al. , 2002).…”

Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE

“…Because yeast Arf1p also contains MXXE, and can be recruited to Golgi when expressed in mammalian cells (unpublished data), this motif may be used for Golgi targeting in yeast cells. Indeed, evidence that the α3 region in yeast promotes Golgi recruitment of Arf-1 comes from a genetic study that identified amino acid residues in the distal portion of helix α3 as an intragenic suppressor of a dominant lethal mutation ( Click et al, 2002 ).…”