Chemical-genetic inhibition of sensitized mutant unconventional myosins.

The construction of a sensitized mutant unconventional myosin is an excellent method for determining the function of the individual myosin against a background of related myosins with partially overlapping functions. In this chapter, we outline the steps involved in sensitizing myosin by mutation and screening them against panels of nucleotide analogs, including transfection, microinjection, and actin co-sedimentation in vitro. We also describe conditions and considerations involved in designing functional experiments after a mutant and cognate analog have been identified. The powerful strategy of chemical genetics, when correctly applied to unconventional myosins, enables both the specific and selectable inhibition of the target motor with outstanding internal controls.

Differential regulation of dynein-driven melanosome movement.

Cytoplasmic dyneins are multisubunit minus-end-directed microtubule motors. Different isoforms of dynein are thought to provide a means for independent movement of different organelles. We investigated the differential regulation of dynein-driven transport of pigment organelles (melanosomes) in Xenopus melanophores. Aggregation of melanosomes to the cell center does not change the localization of mitochondria, nor does dispersion of melanosomes cause a change in the perinuclear localization of the Golgi complex, indicating that melanosomes bear a dedicated form of dynein. We examined the subcellular fractionation behavior of dynein light intermediate chains (LIC) and identified at least three forms immunologically, only one of which fractionated with melanosomes. Melanosome aggregation was specifically blocked after injection of an antibody recognizing this LIC. Our data indicate that melanosome-associated dynein is regulated independently of bulk cytoplasmic dynein and involves a subfraction of dynein with a distinct subunit composition.

Improved sensitivity for phosphopeptide mapping using capillary column HPLC and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins.

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.

Motor-cargo interactions: the key to transport specificity.

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.