Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca(2+)-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks identified a similar sequence in the COOH-terminal region of the alpha-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca(2+)-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca(2+)-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the alpha-subunit of actin capping proteins is biologically significant.

The determination of inulin concentration in nanoliter fluid samples is fundamental to micropuncture investigations of renal function, and this is generally accomplished through the use of radioisotopes. We report here a simple and reliable alternative to the use of radioisotopes that employs FITC-labeled inulin. Samples containing FITC-inulin are stored between oil columns in constant-bore microcapillary tubes, which are then used as cuvettes to determine fluorescence on a microscope fluorometer. Standard curves were generated and found to be linear, with correlation coefficients ( R) exceeding 0.99 in every case. Although the fluorescence of FITC-inulin was found to be pH dependent, the pH and fluorescence of each 20- to 40-nl sample could be normalized by the addition of 1 nl of 0.5 M HEPES at pH 7.5. In mice prepared for standard micropuncture, simultaneous measurements of tubular fluid-to-plasma ratios (TF/P) using FITC-inulin and [125I]iothalamate were highly correlated (slope = 0.95, y-intercept = 0.01, R = 0.942), as were whole kidney measurements of glomerular filtration rate (GFR) (slope = 1.25, y-intercept = −53.5 μl/min, R = 0.99). Micropuncture determinations of late-proximal samples from mice before and after treatment with acetazolamide showed expected changes: TF/P of FITC-inulin decreased from 1.89 ± 0.07 to 1.48 ± 0.10; single-nephron GFR (SNGFR) decreased from 9.64 ± 1.1 to 6.65 ± 1.0 nl/min; and fractional fluid reabsorption decreased from 45.3 ± 1.9 to 26.8 ± 5.2%. Measurements of TF/P of FITC-inulin, volume, and SNGFR using this technique were stable for at least 2 wk when samples were stored in the dark at 4°C. These data demonstrate that this simple method for determining inulin clearance represents a viable and accurate alternative to radioactive methods. This approach has the added benefits of being relatively inexpensive and leaving the micropuncture sample intact.

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A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4% of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4% of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60% of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20% of 3T3 membrane actin and 40% of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.Actin has now been identified as a component of essentially all eukaryotic animal cells in which it has been sought. In addition, myosin-and tropomyosin-like proteins have been identified in many of these nonmuscle cells. (An extensive review on the subject of contractile proteins of nonmuscle cells has recently appeared [29].) In several electron microscope preparations, apparent contact between plasma membrane and actin filaments (or thin filaments with the dimensions of actin) has been observed in situ (24,36,42,44,45) or in isolated plasma membranes (28). Biochemical evidence has also been presented that actin is associated with membranes of synaptosomes (3). It is, therefore, likely that actin filaments function at least in part via attachment to the plasma membrane of cells. Furthermore, control of the membrane-actin association seems a likely point for regulation of actin function as an effector of diverse cellular movements and morphological changes. We have, therefore, chosen as the initial phase of studies on the mechanisms of cellular movements to try to identify actin in membranes from 3T3 mouse fibroblasts and HeLa cells.

Arabinogalactan-proteins (AGPs) have been purified from the plasma membrane of suspension-cultured Paul's Scarlet rose (Rosa sp.) cells. The two most abundant and homogeneous plasma membrane ACP fractions were named plasma membrane ACPl (PM-ACP1) and plasma membrane AGP2 (PM-ACP2) and had apparent molecular masses of 140 and 21 7 kD, respectively. Both PM-ACP1 and PM-ACP2 had p-(l-3)-, p-(1,6)-, and p-(1,3,6)-galactopyrano-syl residues, predominantly terminal a-arabinofuranosyl residues, and (1,4)-and terminal glucuronopyranosyl residues. The protein moieties of PM-ACP1 and PM-ACP2 were both rich in hydroxypro-lhe, alanine, and serine, but differed in the abundance of hy-droxyproline, which was 1.6 times higher in PM-ACP2 than in PM-ACP1. Another difference was the overall protein content, which was 3.7% (w/w) in PM-ACP1 and 15% in PM-ACP2. As judged by their behavior on reverse-phase chromatography, PM-ACPl and PM-ACP2 were not more hydrophobic than ACPs from the cell wall or culture medium. In contrast, a minor plasma membrane ACP fraction eluted later on reverse-phase chromatography and was more negatively charged at pH 5 than either PM-ACP1 or PM-ACP2. The more negatively charged fraction contained molecules with a glycosyl composition characteristic of ACPs and included at least two different macromolecules. The results of this investigation indicate that Rosa plasma membrane contains at least four distinct ACPs or ACP-like molecules. These molecules differed from each other in size, charge, hydrophobicity, amino-acyl composition , and/or protein content. AGPs are a class of proteoglycans that are rich in galac-tosyl and arabinosyl residues (Clarke et al., 1979; Fincher et al., 1983; Bacic et al., 1988), and they appear to be present in all plant cells (Jermyn and Yeow, 1975; Clarke et al.,

Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus. We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity. IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus. Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

Intracellular free calcium ([Ca2+]i) has been proposed to play an important part in the regulation of the cell cycle. Although a number of studies have shown that stimulation of quiescent cells with growth factors causes an immediate rise in [Ca21]i (Rabinovitch et al., 1986;Vincentini and Villereal, 1986;Hesketh et al., 1988;Tucker et al., 1989;Wahl et al., 1990) This study is the first to show a direct relationship between early serum stimulated Cai2+ increase and subsequent DNA synthesis in human cells. It also goes beyond recent studies on BALB/3T3 cells by providing dose response data and demonstrating reversibility, which are strong indications of a cause and effect relationship.