Juliane P. Caviston

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Huntingtin facilitates dynein/dynactin-mediated vesicle transport

Cytoplasmic dynein is a multisubunit microtubule motor complex that, together with its activator, dynactin, drives vesicular cargo toward the minus ends of microtubules. Huntingtin (Htt) is a vesicle-associated protein found in both neuronal and nonneuronal cells that is thought to be involved in vesicular transport. In this study, we demonstrate through yeast two-hybrid and affinity chromatography assays that Htt and dynein intermediate chain interact directly; endogenous Htt and dynein coimmunoprecipitate from mouse brain cytosol. Htt RNAi in HeLa cells results in Golgi disruption, similar to the effects of compromising dynein/dynactin function. In vitro studies reveal that Htt and dynein are both present on vesicles purified from mouse brain. Antibodies to Htt inhibited vesicular transport along microtubules, suggesting that Htt facilitates dynein-mediated vesicle motility. In vivo inhibition of dynein function results in a significant redistribution of Htt to the cell periphery, suggesting that dynein transports Htt-associated vesicles toward the cell center. Together these findings indicate that Htt binds to dynein and acts in a complex along with dynactin and Htt-associated protein-1 to facilitate vesicular transport.

Huntingtin as an essential integrator of intracellular vesicular trafficking.

The neurodegenerative disorder Huntingtons disease is caused by an expansion in the polyglutamine repeat region of the protein huntingtin. Multiple studies in cellular and animal model systems indicate that this mutation imparts a novel toxic function required for disease pathogenesis. However, the normal function of huntingtin, an essential cellular protein in higher vertebrates, is not yet well understood. Emerging data indicate an important role for wild-type huntingtin in the intracellular transport of vesicles and organelles. Here, we discuss current progress on the role of huntingtin in vesicular trafficking, focusing on the proposal that huntingtin might be a crucial regulator of organelle transport along the cellular cytoskeleton.

A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

We investigated the role of the membrane-associated scaffolding protein huntingtin (Htt) in the dynein-mediated transport of early, recycling, and late endosomes and lysosomes. Our observations support a model of Htt as a facilitator of dynein-mediated trafficking that can regulate the cytoskeletal association of dynamic organelles.

Singularity in budding: A role for the evolutionarily conserved small GTPase Cdc42p

The budding yeast Saccharomyces cerevisiae initiates polarized growth or budding once per cell cycle at a specific time of the cell cycle and at a specific location on the cell surface. Little is known about the molecular nature of the temporal and spatial regulatory mechanisms. It is also unclear what factors, if any, among the numerous proteins required to make a bud are involved in the determination of budding frequency. Here we describe a class of cdc42 mutants that produce multiple buds at random locations on the cell surface within one nuclear cycle. The critical mutation responsible for this phenotype affects amino acid residue 60, which is located in a domain required for GTP binding and hydrolysis. This mutation bypasses the requirement for the essential guanine-nucleotide-exchange factor Cdc24p, suggesting that the alteration at residue 60 makes Cdc42p hyperactive, which was confirmed biochemically. This result also suggests that the only essential function of Cdc24p is to activate Cdc42p. Together, these data suggest that the temporal and spatial regulation of polarized growth converges at the level of Cdc42p and that the activity of Cdc42p determines the budding frequency.