Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions. Molecular & Cellular

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the coimmunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca 2؉ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca 2؉ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca 2؉ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18 -46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca 2؉ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC 50 of 300 M). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca 2؉ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca 2؉ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.

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