Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Eukaryotic cells compartmentalize biochemical reactions into membrane‐enclosed organelles that must be faithfully propagated from one cell generation to the next. Transport and retention processes balance the partitioning of organelles between mother and daughter cells. Here we report the identification of an ER‐peroxisome tether that links peroxisomes to the ER and ensures peroxisome population control in the yeast Saccharomyces cerevisiae. The tether consists of the peroxisome biogenic protein, Pex3p, and the peroxisome inheritance factor, Inp1p. Inp1p bridges the two compartments by acting as a molecular hinge between ER‐bound Pex3p and peroxisomal Pex3p. Asymmetric peroxisome division leads to the formation of Inp1p‐containing anchored peroxisomes and Inp1p‐deficient mobile peroxisomes that segregate to the bud. While peroxisomes in mother cells are not released from tethering, de novo formation of tethers in the bud assists in the directionality of peroxisome transfer. Peroxisomes are thus stably maintained over generations of cells through their continued interaction with tethers.

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Calreticulin is an endoplasmic reticulum (ER) luminalmutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.The endoplasmic reticulum (ER) 1 plays an essential role in a variety of cellular processes, including Ca 2ϩ homeostasis, protein and lipid synthesis, and post-translational modification and folding of membrane-associated and secreted proteins (1). The ER ensures that only correctly folded proteins proceed through the secretory pathway and directs misfolded proteins to ER-associated degradation (2, 3). The lumen of the ER is a dynamic environment that contains numerous molecular chaperones and Ca 2ϩ -binding proteins that are designed for these tasks. Molecular chaperones are proteins that bind to misfolded/unfolded proteins in a transient manner to assist in their folding.Calreticulin is a Ca 2ϩ -binding chaperone that resides in the lumen of the ER and is involved in modulation of Ca 2ϩ homeostasis and in the folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle" (4 -7). Calreticulin and calnexin are both ER lectins, which bind transiently to virtually all newly synthesized glycoproteins (5-7). Chaperone-assisted protein folding has been studied extensively using Escherichia coli GroEL heat shock proteins, which are cytoplasmic (8). Numerous studies have been carried out on ER-associated chaperones (2-7); yet, the molecular features of calreticulin that confer its chaperone function have not yet been determined (7).Three distinct structural domains have been identified in calreticulin: the amino-terminal, globular N-domain; the central P-domain; and the carboxyl-terminal C-domain (7). NMR (9), modeling (10), and biochemical studies (11) indicate that the globular N-domain and the "extended arm" P-domain of calreticulin may form a functional protein-folding unit (10). This region of calreticulin contains a Zn 2ϩ binding site and one disulfide bond, and it may also bind ATP (12)(13)(14). When calreticulin binds Zn 2ϩ , it undergoes dramatic conformational changes (15). Chemical modification of calreticulin has revealed that four histidines located in the N-domain of the protein (His 25 , His 82 , His 128 , and His 153 ) are involved in the Zn 2ϩ binding (12). The Zn 2ϩ -dependent conformational change in calreticulin affects its ability to bind to unfolded protein/ glycoprotein substrates in vitro (16), suggesting that conformational changes in calreticulin may modify its chaperone function. The role of the Zn 2ϩ binding histidine residues in calreticulin function is not known.Calreticulin deficiency is embryonic lethal, and cells derived from calreticulin knockout embryos have impaired Ca 2ϩ homeostasis and compromised protein folding and quality control (11,17). The availability of calret...

Using a proteomic analysis of the luminal environment of the endoplasmic reticulum (ER), we have identified 141 proteins, of which 6 were previously unknown. The endoplasmic reticulum (ER)1 is a centrally located intracellular organelle involved in protein and lipid synthesis and Ca 2ϩ storage and release (1). Disruption of ER homeostasis results in organellar disease with detrimental effects at both cellular and systemic levels including metabolic, developmental, and neurodegenerative conditions and protein folding disorders (2-5). This is not surprising, as more than 30% of all proteins are synthesized in the ER before being distributed to other locations in the cell. Many Ca 2ϩ -binding chaperones reside in the lumen of the ER and are involved in virtually every aspect of ER function, including the regulation of Ca 2ϩ homeostasis, the folding, oligomerization, and glycosylation of proteins, and the formation and isomerization of disulphide bonds within proteins (6, 7). While the complexity of ER function is recognized, the protein composition of the ER and its luminal environment remains to be fully analyzed.The analysis of complex protein mixtures has recently been facilitated by the development of two-dimensional (2D) gel electrophoresis with immobilized pH gradient strips, allowing for the separation of hundreds of proteins on a single gel. In addition, mass spectrometry (MS) provides a sensitive analytical tool that allows for the identification of very small amounts of individual proteins. These techniques have been combined successfully to determine the protein compositions of subcellular structures (8 -10) and specialized cell types (11). This study emphasizes the luminal proteome of mouse liver ER. A 2D protein map of the ER luminal environment, which included over 2,000 spots, was generated. Peptide analyses by matrix-assisted laser desorption/ionization mass spectroscopy and tandem mass spectrometry (MALDI-MS/MS) allowed unambiguous identification of more than 140 different proteins, including several that were not previously known. Two of these "new" luminal ER proteins, ERp19 and ERp46, contain thioredoxin motifs that are typically found in PDI-like proteins. Functional studies revealed that ERp46 but not ERp19 can compensate for the loss of PDI function in yeast. This work clearly illustrates and deepens our understanding of the complexity of the ER lumen and suggests a multiplicity of PDI family members. EXPERIMENTAL PROCEDURESFractionation and Characterization of the Liver Membrane-ER vesicles, Golgi apparatus, plasma membrane, nuclei, and mitochondria were purified from livers of Balb/C mice (12) with some modifications to accommodate 2D gel electrophoresis requirements. ER vesicles were centrifuged twice on discontinuous sucrose gradients followed by two washes with 10 mM Tris-HCl, pH 7.5. One-mg aliquots of the isolated ER vesicles were pelleted by centrifugation at 90,000 ϫ g for 60 min and stored at Ϫ80°C until use.For electron microscopy analysis, an aliquot of freshly prepared ER vesicl...

Reversible phosphorylation is the most common posttranslational modification used in the regulation of cellular processes. This study of phosphatases and kinases required for peroxisome biogenesis is the first genome-wide analysis of phosphorylation events controlling organelle biogenesis. We evaluate signaling molecule deletion strains of the yeast Saccharomyces cerevisiae for presence of a green fluorescent protein chimera of peroxisomal thiolase, formation of peroxisomes, and peroxisome functionality. We find that distinct signaling networks involving glucose-mediated gene repression, derepression, oleate-mediated induction, and peroxisome formation promote stages of the biogenesis pathway. Additionally, separate classes of signaling proteins are responsible for the regulation of peroxisome number and size. These signaling networks specify the requirements of early and late events of peroxisome biogenesis. Among the numerous signaling proteins involved, Pho85p is exceptional, with functional involvements in both gene expression and peroxisome formation. Our study represents the first global study of signaling networks regulating the biogenesis of an organelle.

In Saccharomyces cerevisiae, the class V myosin motor Myo2p propels the movement of most organelles. We recently identified Inp2p as the peroxisome-specific receptor for Myo2p. In this study, we delineate the region of Myo2p devoted to binding peroxisomes. Using mutants of Myo2p specifically impaired in peroxisome binding, we dissect cell cycle–dependent and peroxisome partitioning–dependent mechanisms of Inp2p regulation. We find that although total Inp2p levels oscillate with the cell cycle, Inp2p levels on individual peroxisomes are controlled by peroxisome inheritance, as Inp2p aberrantly accumulates and decorates all peroxisomes in mother cells when peroxisome partitioning is abolished. We also find that Inp2p is a phosphoprotein whose level of phosphorylation is coupled to the cell cycle irrespective of peroxisome positioning in the cell. Our findings demonstrate that both organelle positioning and cell cycle progression control the levels of organelle-specific receptors for molecular motors to ultimately achieve an equidistribution of compartments between mother and daughter cells.

Peroxisomes are dynamic organelles that divide continuously in growing cell cultures and expand extensively in lipid-rich medium. Peroxisome population control is achieved in part by Pex11p-dependent regulation of peroxisome size and number. Although the production of Pex11p in yeast is tightly linked to peroxisome biogenesis by transcriptional regulation of the PEX11 gene, it remains unclear if and how Pex11p activity could be modulated by rapid signaling. We report the reversible phosphorylation of Saccharomyces cerevisiae Pex11p in response to nutritional cues and delineate a mechanism for phosphorylation-dependent activation of Pex11p through the analysis of phosphomimicking mutants. Peroxisomal phenotypes in the PEX11-A and PEX11-D strains expressing constitutively dephosphorylated and phosphorylated forms of Pex11p resemble those of PEX11 gene knock-out and overexpression mutants, although PEX11 transcript and Pex11 protein levels remain unchanged. We demonstrate functional inequality and differences in subcellular localization of the Pex11p forms. Pex11Dp promotes peroxisome fragmentation when reexpressed in cells containing induced peroxisomes. Pex11p translocates between endoplasmic reticulum and peroxisomes in a phosphorylationdependent manner, whereas Pex11Ap and Pex11Dp are impaired in trafficking and constitutively associated with mature and proliferating peroxisomes, respectively. Overexpression of cyclin-dependent kinase Pho85p results in hyperphosphorylation of Pex11p and peroxisome proliferation. This study provides the first evidence for control of peroxisome dynamics by phosphorylation-dependent regulation of a peroxin.Peroxisomes are a group of organelles characterized by high metabolic plasticity that act in a variety of important biochemical processes, notably the metabolism of lipids and the detoxification of reactive oxygen species. Although functional peroxisomes are essential for human survival, the conditional viability of yeast peroxisome biogenesis mutants has enabled the molecular identification of the core biogenic components of this organelle (1). Peroxisome biogenesis can be viewed as a complex developmental program that is initiated by the fatty acid-induced expression of many genes coding for peroxisomal proteins (2), followed by the stepwise assembly of the organelle. This highly regulated process can be divided into early and late events that are distinguished by the formation of preperoxisomal Pex3p-containing vesicles from the endoplasmic reticulum (ER) 2 (3, 4) and the entry of peroxisomal enzymes from the cytosol to form metabolically active peroxisomes. Different receptor-mediated import pathways for matrix (5) and membrane (6) proteins as well as a succession of distinct precursor populations (7) contribute to the orderly assembly of peroxisomes.Peroxisome population control is accomplished by balancing peroxisome formation, division, and turnover and peroxisome partitioning to daughter cells (8). The Pex11 family of peroxisomal membrane proteins (PMPs) regulates peroxi...