Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

The influence of water depth and flow regime on phytoplankton biomass and community structure in a shallow,lowland river

Hydrobiologia - The taxonomic composition and biomass of phytoplankton in the San Joaquin River, California, were examined in relation to water depth, flow regime, and water chemistry. Without...     

Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells

Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.     

Specific regional differences of in vitro beta-endorphin metabolism in schizophrenics

Incubation of beta-endorphin (beta-E; 25 microM) with twice-washed brain membrane homogenates leads to the formation of several biologically active peptide fragments which have been shown to be present in the brain. Based on clinical studies, some of these endorphin fragments have been shown to be active in patients with neuropsychiatric disease states. We studied the regional specificity of beta-E metabolism in frontal cortex versus putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that cortical tissue has a lower rate of gamma-endorphin production from beta-E and a similar rate of des-tyrosine-gamma-endorphin production. Significant differences were noted in the production of other active fragments (beta-E (1-16, 2-16, 6-21)). These results support the hypothesis that there is a regional specificity of beta-E metabolism in the brain, and these differences may have important functional consequences to secreted peptides and important clinical consequences in schizophrenia.