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In yeast, the transition between the fermentative and the oxidative metabolism, called the diauxic shift, is associated with major changes in gene expression and protein synthesis. The zinc cluster protein Cat8p is required for the derepression of nine genes under nonfermentative growth conditions (ACS1, FBP1, ICL1, IDP2, JEN1, MLS1, PCK1, SFC1, and SIP4). To investigate whether the transcriptional control mediated by Cat8p can be extended to other genes and whether this control is the main control for the changes in the synthesis of the respective proteins during the adaptation to growth on ethanol, we analyzed the transcriptome and the proteome of a cat8⌬ strain during the diauxic shift. In this report, we demonstrate that, in addition to the nine genes known as Cat8p-dependent, there are 25 other genes or open reading frames whose expression at the diauxic shift is altered in the absence of Cat8p. For all of the genes characterized here, the Cat8p-dependent control results in a parallel alteration in mRNA and protein synthesis. It appears that the biochemical functions of the proteins encoded by Cat8p-dependent genes are essentially related to the first steps of ethanol utilization, the glyoxylate cycle, and gluconeogenesis. Interestingly, no function involved in the tricarboxylic cycle and the oxidative phosphorylation seems to be controlled by Cat8p.

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Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.Water is the universal solvent and most important molecule for life. Immense water volumes pass across the membranes of all living cells, especially across the plasma membranes of plants (1). Simple diffusion of water through the lipid bilayer has an activation energy of Ͼ10 kcal/mol and cannot explain the rapid water flow through human red cell membranes found to exhibit an activation energy of Ͻ5 kcal/mol. This led to the hypothesis that water pores must exist (2). The discovery (3) and cloning (4) of the first known water channel, aquaporin-1 (AQP1), 1 from human erythrocytes and the demonstration of water transport in Xenopus oocytes expressing its complementary RNA (5) confirmed this hypothesis.Since then, a large number of channel-forming integral proteins homologous to AQP1 have been found in all forms of life (6). This membrane protein family was initially named the MIP family after its first sequenced member, the major intrinsic protein (MIP) of bovine lens fiber cells (7). Multiple sequence alignment and phylogenetic analysis of 164 members of the MIP family, now frequently referred to as aquaporin super family, revealed 16 subfamilies that form two distinct clusters, the aquaporin (AQP) cluster and the glycerol facilitator-like cluster (8). The AQPs are highly specific for water, whereas the glycerol facilitators allow the passage of small, nonionic molecules such as glycerol and urea (9). In addition, ovine MIP tetramers have been found to form a groove and tongue contact via their extracellular surfaces, lending support to a dual function of the protein, as a water channel and as a cell to cell adhesion molecule in the eye lens (10).Most members of the aquaporin super family...

DNA replication in Plasmodium parasites takes place at multiple distinct points during their complex life cycle in the mosquito and vertebrate hosts. Although several parasite proteins involved in DNA replication have been described, the various mechanisms engaged in DNA metabolism of this major pathogen remain largely unexplored. As a step toward understanding this complex network, we describe the identification of Plasmodium falciparum replication protein A large subunit (pfRPA1) through affinity purification and mass spectral analysis of a purified 55-kDa factor. Gel retardation experiments revealed that pfRPA is the major singlestranded DNA binding activity in parasite protein extracts. The activity was expressed in a cell cycle-dependent manner with peak activities in late trophozoites and schizonts, thus correlating with the beginning of chromosomal DNA replication. Accordingly, the pfrpa1 message was detected in parasites 20 -24 h post-invasion which is in agreement with the expression of other P. falciparum DNA replication genes. Our results show that pfRPA1 is encoded by an unusual 6.5-kb transcript containing a single open reading frame of which only the C-terminal 42% of the deduced protein sequence shows homologies to other reported RPA1s. Like the orthologues of other protozoan parasites, pfRPA1 lacks the N-terminal protein interaction domain and is thus remarkably smaller than the RPA1s of higher eukaryotes.Plasmodium falciparum causes one of the most life-threatening parasitic diseases in humans being responsible for up to 2 million deaths per year. Malaria pathogenesis is associated with the intracellular erythrocytic stage of the life cycle of the parasite involving repeated rounds of invasion, growth, and schizogony. Parasites that eventually differentiate into gametocytes are taken up by the female anopheline vector where zygote formation and sporogony take place. Sporozoites injected into a human host by the bite of an infective mosquito invade hepatocytes and, after schizogony, release thousands of merozoites capable of invading red blood cells. During each replication event, the timing, rate, and extent of genome multiplication have to be controlled appropriately and coordinated at each developmental stage. Most of the studies on DNA replication in P. falciparum have been conducted during the erythrocytic stages. Several genes involved in eukaryotic chromosomal DNA replication and their encoded proteins have been identified in this parasite, including DNA polymerases ␣ (DNA pol ␣) 1 (1, 2) and ␦ (DNA pol ␦) (3, 4), proliferating cell nuclear antigen (5), and topoisomerases I (6) and II (7). It has been shown that expression of these genes follows a stagespecific pattern coinciding with the beginning of chromosomal replication which starts 28 -31 h after merozoite invasion and continues through most of schizogony (8).Due to their complex life cycle and constant immunological pressure exerted by their hosts, the processes of DNA metabolism in Plasmodium parasites must be both very efficient a...

The protein kinase TOR (target of rapamycin) controls several steps of ribosome biogenesis, including gene expression of rRNA and ribosomal proteins, and processing of the 35S rRNA precursor, in the budding yeast Saccharomyces cerevisiae. Here we show that TOR also regulates late stages of ribosome maturation in the nucleoplasm via the nuclear GTP‐binding protein Nog1. Nog1 formed a complex that included 60S ribosomal proteins and pre‐ribosomal proteins Nop7 and Rlp24. The Nog1 complex shuttled between the nucleolus and the nucleoplasm for ribosome biogenesis, but it was tethered to the nucleolus by both nutrient depletion and TOR inactivation, causing cessation of the late stages of ribosome biogenesis. Furthermore, after this, Nog1 and Nop7 proteins were lost, leading to complete cessation of ribosome maturation. Thus, the Nog1 complex is a critical regulator of ribosome biogenesis mediated by TOR. This is the first description of a physiological regulation of nucleolus‐to‐nucleoplasm translocation of pre‐ribosome complexes.