Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Insulin-secreting pancreatic beta cells are exceptionally rich in zinc. In these cells, zinc is required for zinc-insulin crystallization within secretory vesicles. Secreted zinc has also been proposed to be a paracrine and autocrine modulator of glucagon and insulin secretion in pancreatic alpha and beta cells, respectively. However, little is known about the molecular mechanisms underlying zinc accumulation in insulin-containing vesicles. We previously identified a pancreas-specific zinc transporter, ZnT-8, which colocalized with insulin in cultured beta cells. In this paper we studied its localization in human pancreatic islet cells, and its effect on cellular zinc content and insulin secretion. In human pancreatic islet cells, ZnT-8 was exclusively expressed in insulin-producing beta cells, and colocalized with insulin in these cells. ZnT-8 overexpression stimulated zinc accumulation and increased total intracellular zinc in insulin-secreting INS-1E cells. Furthermore, ZnT-8-overexpressing cells display enhanced glucose-stimulated insulin secretion compared with control cells, only for a high glucose challenge, i.e. >10 mM glucose. Altogether, these data strongly suggest that the zinc transporter ZnT-8 is a key protein for both zinc accumulation and regulation of insulin secretion in pancreatic beta cells.

We report here that the negative cell cycle regulator protein p53 is an in vivo and in vitro substrate for protein kinase C, a cellular receptor for the tumor-promoter phorbol esters. We also demonstrate that p53 interacts in a calcium-dependent manner with SlOOb, a member of the S100 protein family involved in cell cycle progression and cell differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and SlOOb was utilized for the purification of cellular and recombinant murine p53 by affinity chromatography with SlOOb-Sepharose. Furthermore, and of particular interest, we have shown that purified p53 undergoes temperaturedependent oligomerization and that the interaction between SlOOb and p53 not only induces total inhibition of p53 oligomerization but also promotes disassembly of the p53 oligomers. We suggest that these effects result from the binding of SlOOb to the multifunctional basic C-terminal domain of p53 and propose that p53 may be a cellular target for the S100 protein family members involved in the control of the cell cycle at the

A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS͞PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS͞MS data, a BLAST-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple ␣-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems. P lastids, and especially chloroplasts, conduct vital biosynthetic functions, and many reactions are located exclusively within these unique organelles. A two-membrane system, the envelope, surrounds all plastid types and separates the plastid stroma from the cytosol. As a consequence, the envelope is involved in the controlled exchange of a variety of ions and metabolites between these two subcellular compartments (1).Chloroplasts import cytoplasmically synthesized precursor proteins from the cytosol. Translocation of precursor proteins across the envelope is achieved by the joint action of Toc and Tic translocons located at the outer and inner envelope membranes, respectively, of the chloroplast envelope (2, 3). Chloroplasts also take up intermediates of various metabolic pathways such as dicarboxylic acids, acetate, and phosphoenolpyruvate. Chloroplasts also have been demonstrated to import inorganic ions like K ϩ , Na (4, 5). As the sole site of biosynthesis of most amino acids (with the exception of sulfur-containing amino acids; refs. 6 and 7), chloroplasts must export these compounds for protein synthesis in the cytosolic and mitochondrial compartments. Finally, because of metabolism compartmentation, several other organic or inorganic comp...

Ml clone S6 myeloid leukemic cells do not express detectable p53 protein. When stable transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in Ml cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G, appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated Ml cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growthregulatory signals.The p53 phosphoprotein is the product of a tumor suppressor gene, whose inactivation may play a role in the development and progression of many types of cancer (reviewed in references 5, 26, 35, and 43). In most cases,

Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P 1B -type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.Chloroplasts contain a large variety of ions among which metal ions such as copper, iron, manganese, and zinc that are essential for their development and function. Copper is an essential redox cofactor required for a wide variety of processes, including photosynthetic electron transfer reactions (plastocyanin) and detoxification of superoxide radicals (Cu/Zn-superoxide dismutase, SOD) 4 (1). Other metal ions are cofactors for several enzymatic reactions: zinc is associated with chloroplast SOD, methionine synthase, carbonic anhydrase; manganese is required for oxygen evolution in photosynthesis; whereas iron is a cofactor of iron SOD and is found in iron-sulfur clusters of cytochrome b 6 f complex, ferredoxin, photosystem I (PSI), and photosystem II (PSII) (2). All these metals are essential micronutrients but are toxic when present in excess (3). To maintain the concentration of metals within physiological limits, cells possess mechanisms that control the uptake, accumulation, trafficking, and also detoxification of metal ions. Little is known about metal transport into chloroplasts. Until now, the sole chloroplast proteins demonstrated as being involved in metal ions transport are PAA1 (4) and very recently PAA2 (5), two P 1B -type ATPases. PAA1, localized into the chloroplast envelope, supplies copper to the chloroplast, whereas PAA2, localized into the thylakoid membrane, delivers copper to the thylakoid lumen. One important result derived from this work is the fact that the disruption of the PAA1 gene does not fully abolish the import of copper into the chloroplast. From this recent observation, Abdel-Ghany and coworkers (5) concluded that an as yet unidentified and additional way to PAA1 must exist to import copper into the chloroplast....

The large majority of plastid proteins are nuclearencoded and, thus, must be imported within these organelles. Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region. In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope. Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane. Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein. These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein. These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence.Higher plant plastids contain a genome with limited coding capacity. The large majority of plastid proteins are nuclearencoded and thus, must be imported within these organelles. To this purpose, the two envelope membranes that surround chloroplasts contain a protein import apparatus constituted of the TOC and TIC complexes (translocon at the inner or outer membranes of the chloroplast envelope) (for recent reviews, see Refs. 1-4). The major element of this translocation complex is Toc75, which is the most abundant protein in the outer envelope membrane. Moreover, Toc75 seems to form the central pore of the outer envelope translocation channel (5, 6) and interacts specifically with the N-terminal transit peptide of precursor proteins during the import process toward chloroplast (7). This cleavable and N-terminal transit peptide was shown to be necessary and sufficient for transport of precursors (i) across the two envelope membranes (8, 9) and (ii) across the thylakoid membrane if the transit sequence is bipartite (it contains additional targeting information for thylakoid lumen targeting) (9). The cleavage of the transit sequence is performed by two proteases, one in the stroma (10) and one in the thylakoid lumen for the bipartite transit sequences (11). Moreover it is interesting to note that all described inner membrane proteins and intermembrane space proteins possess an N-terminal cleavable transit peptide, while most outer envelope membrane proteins do not have any cleavable transit peptide. In the latter case, the targeting information is contained within the mature protein (12), and after their cytosolic synthesis, proteins are directly incorporated in the lipid bilayer th...

Late-embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to a high level in desiccation-tolerant tissues and are thus prominent in seeds. They are expected to play a protective role during dehydration; however, functional evidence is scarce. We identified a LEA protein of group 3 (PsLEAm) that was localized within the matrix space of pea (Pisum sativum) seed mitochondria. PsLEAm revealed typical LEA features such as high hydrophilicity and repeated motifs, except for the N-terminal transit peptide. Most of the highly charged protein was predicted to fold into amphiphilic a-helixes. PsLEAm was expressed during late seed development and remained in the dry seed and throughout germination. Application of the stress hormone abscisic acid was found to reinduce the expression of PsLEAm transcripts during germination. PsLEAm could not be detected in vegetative tissues; however, its expression could be reinduced in leaves by severe water stress. The recombinant PsLEAm was shown to protect two mitochondrial matrix enzymes, fumarase and rhodanese, during drying in an in vitro assay. The overall results constitute, to our knowledge, the first characterization of a LEA protein in mitochondria and experimental evidence for a beneficial role of a LEA protein with respect to proteins during desiccation.Late-embryogenesis abundant (LEA) proteins are overwhelmingly hydrophilic proteins that accumulate to high levels in the latter stages of seed maturation and disappear following germination (Galau et al., 1986). While almost ubiquitous in the plant kingdom, data mining has revealed the widespread occurrence of LEA proteins in prokaryotes and eucaryotes (GarayArroyo et al., 2000). Historically clustered in five main groups based on primary structure analysis (Dure et al., 1989;Cuming, 1999), the LEA protein classification was recently reexamined using statistically based bioinformatic tools (Wise, 2003).LEA protein expression, which often appears abscisic acid (ABA) dependent, can also occur in vegetative tissues subjected to water deficit associated with drought, salt, or cold stress (for review, see Ingram and Bartels, 1996;Thomashow, 1998;Cuming, 1999). Both the pattern of expression and the structural features of LEA proteins suggest a general protective role in desiccation tolerance (Ingram and Bartels, 1996;Cuming, 1999). This hypothesis was recently supported by the discovery of a LEA protein in an anhydrobiotic nematode (Browne et al., 2002) as well as by the sensitization to desiccation induced by mutational inactivation of LEA genes in the prokaryote Deinococcus radiodurans (Battista et al., 2001). In view of the apparent lack of well-ordered tertiary structure of LEA proteins preventing their use as catalysts, several mechanisms have been proposed to relate their structural features to the protection of cellular structures required by a dehydrated state: water replacement, ion sequestering, macromolecules, and membrane stabilization (Close, 1996(Close, , 1997Cuming, 1999). Experimentally, sev...