Divalent cations regulate connexin hemichannels by modulating intrinsic voltage-dependent gating

Connexin hemichannels are robustly regulated by voltage and divalent cations. The basis of voltage-dependent gating, however, has been questioned with reports that it is not intrinsic to hemichannels, but rather is derived from divalent cations acting as gating particles that block the pore in a voltage-dependent manner. Previously, we showed that connexin hemichannels possess two types of voltage-dependent gating, termed V_j and loop gating, that in Cx46 operate at opposite voltage polarities, positive and negative, respectively. Using recordings of single Cx46 hemichannels, we found both forms of gating persist in solutions containing no added Mg²⁺ and EGTA to chelate Ca²⁺. Although loop gating persists, it is significantly modulated by changing levels of extracellular divalent cations. When extracellular divalent cation concentrations are low, large hyperpolarizing voltages, exceeding −100 mV, could still drive Cx46 hemichannels toward closure. However, gating is characterized by continuous flickering of the unitary current interrupted by occasional, brief sojourns to a quiet closed state. Addition of extracellular divalent cations, in this case Mg²⁺, results in long-lived residence in a quiet closed state, suggesting that hyperpolarization drives the hemichannel to close, perhaps by initiating movements in the extracellular loops, and that divalent cations stabilize the fully closed conformation. Using excised patches, we found that divalent cations are only effective from the extracellular side, indicative that the binding site is not cytoplasmic or in the pore, but rather extracellular. V_j gating remains essentially unaffected by changing levels of extracellular divalent cations. Thus, we demonstrate that both forms of voltage dependence are intrinsic gating mechanisms in Cx46 hemichannels and that the action of external divalent cations is to selectively modulate loop gating.     

The synthesis of export-defective proteins can interfere with normal protein export in Escherichia coli

We have analyzed the kinetics of maturation for certain bacterial envelope proteins in Escherichia coli strains that are also concomitantly producing an export-defective protein. Our data indicate that proteins with defective signal peptides, rendered nonfunctional by either point mutation or deletion, interfere with the normal export of other envelope proteins. Expression of interference requires that the interfering protein: (i) exhibit a major export defect; (ii) be synthesized at a high rate; and (iii) be actively synthesized at the time interference is being measured. The latter data suggest that interference is a cotranslational process. Intragenic or extragenic suppression of the export defect exhibited by the interfering protein relieves interference in a manner that is directly related to strength of suppression. These and additional data suggest that interference occurs at a very early step in the secretory process. We interpret these results to indicate that proteins with defective signal peptides are still recognized as proteins destined for secretion and are, therefore, at least transiently incorporated into the cell's secretory pathway. The incorporation of an export-defective protein into the secretory pathway disrupts the normal protein traffic from the cytoplasm to the various extracellular compartments.     

Functional studies of the mammalian Sac1 phosphoinositide phosphatase

The Sac1 PIP phosphatase is an enigmatic enzyme in that it occupies an intracellular location (ER) that is not normally associated with PIP signaling. Yet, genetic experiments in mice and silencing experiments in cultured cells report an essential housekeeping function for this protein. Detailed cellular analyses report maintenance of proper organization of the Golgi system, and of the mitotic spindle apparatus, are compromised when Sac1 functional thresholds are breached. While the Golgi derangements do not obviously affect protein transport through the organelle, the mitotic defects result in defects in progression through the G2/M stage of the cell cycle. Finally, both the catalytic PIP phosphatase activity, and its ability to be recycled back to the ER, represent essential functional features of the Sac1 enzyme.We expect that current insights for Sac1 will set the blueprint for future analyses of its functions. Many questions remain to be answered in this field: does Sac1 have important roles in ER and plasma membranes connections since ySac1 only degrades the plasma membrane-localized PtdIns 4-OH kinase Stt4 generated PtdIns-4-P? Does the ER-localized Sac1 play critical roles in regulating nuclear PIP signaling since ER is continuous with the nuclear envelope, and does it specifically happen in a certain stage during cell cycle progression? Does anchoring the Sac1-catalytic domain to other cellular membranes affect its function and what effects will be generated by mislocalizing the Sac1-catalytic domain to exotic membrane locations? Does the Golgi dispersion phenotype herald a crosstalk of the organelle with the mitotic apparatus (i.e. does Golgi disorganization provide ectopic nucleation sites for the γ-tubulin ring complex)? Clearly, there is much to be learned regarding the biological functions of Sac1-like lipid phosphatases, and we anticipate the discoveries yet to come will rival those derived from studies of the kinases – both in impact and in scope.     

Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the beta-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of beta-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the beta-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 +/- 0.2% (n = 6) of the cells in the pancreas of newborn (P1) and 0.9 +/- 0.1% (n = 5) of 8-wk-old mice were beta-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying beta-cell biology in normal and diabetic animals.     

Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease

We have mapped a sequence determinant in the vacuolar glycoprotein carboxypeptidase Y (CPY) that directs intracellular sorting of this enzyme. Through the study of hybrid proteins, consisting of amino-terminal segments of CPY fused to the secretory enzyme invertase, we have found that the N-terminal 50 amino acids of CPY are sufficient to direct delivery of a CPY-Inv hybrid protein to the yeast vacuole. Our data suggest that this 50 amino acid segment of CPY contains two distinct functional domains; an N-terminal signal peptide followed by a segment of 30 amino acids that contains the vacuolar sorting signal. Deletion of this putative vacuole sorting signal from an otherwise wild-type CPY protein leads to missorting of CPY. Furthermore, examination of the Asn-linked oligosaccharides present on CPY and CPY-Inv hybrid proteins suggests that an additional determinant in CPY specifies the extent to which these proteins are glycosylated in the Golgi complex.     

Characterization of a region in mature LamB protein that interacts with a component of the export machinery of Escherichia coli

It has been shown that the synthesis of an export-defective protein can interfere with the normal export process in Escherichia coli by limiting the availability of SecB protein, a component of the export apparatus (Collier, D.N., Bankaitis, V.A., Weiss, J.B., and Bassford, P.J. (1988) Cell 53, 273-283). Consistent with this observation, we find that the interference elicited by an export-defective LamB protein is a titratable response resulting from the limitation of a single ligand. We have mapped the interfering region in LamB to between amino acids 320 and 380 of the mature protein. Expression of this sequence in the form of a LacZ-LamB-LacZ fusion protein elicits the export interference phenotype. Deletion of the sequence from an export-defective LamB protein eliminates the ability of this protein to interfere with the export of other secreted proteins. Together, these findings show that this sequence is both necessary and sufficient to cause export interference. Surprisingly, deletion of this sequence from an otherwise wild-type LamB protein does not cause the mutant LamB product to exhibit any obvious export defect. Based on our results, we propose that SecB interacts with both amino acids 320-380 of mature LamB and the LamB signal sequence during initiation of the export process.     

Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex. That some conservation of SEC14p function may exist was initially suggested by experiments that revealed immunoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. We have cloned and characterized the K. lactis SEC14 gene (SEC14KL). Immunoprecipitation experiments indicated that SEC14KL encoded the K. lactis structural homolog of SEC14p. In agreement with those results, nucleotide sequence analysis of SEC14KL revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p. Moreover, a single ectopic copy of SEC14KL was sufficient to render S. cerevisiae sec14-1(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization. This efficient complementation of sec14-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis. Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well. On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported.