A Charge Swap mutation E461K in the yeast dynamin Vps1 reduces endocytic invagination

Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

“…The simplest of these is that Vps1 is recruited to endocytic sites just prior to invagination. The effect of some Vps1 mutants, causing an early defect in the ability of the membrane to invaginate rather than just affecting the postinvagination scission stage, supports this time of arrival ( 14 , 38 ). Following recruitment, Vps1 assembles into rings and higher-order structures.…”

“…Given that endocytic behavior of patches is compromised in the mutant cells, it was relevant to determine whether these phosphomutant Vps1 proteins are able to localize to appropriate cortical sites. We and others have noted that GFP-tagged Vps1 is not fully functional, and we proposed that a likely reason for this is that Vps1, like other dynamins, forms a ring structure, which is unlikely to accommodate 100% tagged protein ( 38 , 44 ). Thus, while a fluorescent tag can be used to indicate localization of Vps1, the lifetime and behavior of the protein are most likely compromised.…”

“…In order to gain further insight into the effect of Vps1 S599 mutants on protein function, mutant forms of the protein were purified. Recently, we were able to demonstrate that purified Vps1 protein can form oligomeric ring structures ( 38 ). The two Vps1 S599 mutants were purified and examined using transmission electron microscopy following negative staining.…”

“…The constructs were then verified by sequencing. Point mutations were introduced into the genome by allele exchange as described previously and verified by sequencing ( 38 ). The bimolecular fluorescence complementation (BiFC) assay used strains carrying Venus constructs crossed for coexpression of both N- and C-terminal halves of Venus.…”