Making the connection: How membrane contact sites have changed our view of organelle biology.

The view of organelles and how they operate together has changed dramatically over the last two decades. The textbook view of organelles was that they operated largely independently and were connected by vesicular trafficking and the diffusion of signals through the cytoplasm. We now know that all organelles make functional close contacts with one another, often called membrane contact sites. The study of these sites has moved to center stage in cell biology as it has become clear that they play critical roles in healthy and developing cells and during cell stress and disease states. Contact sites have important roles in intracellular signaling, lipid metabolism, motor-protein-mediated membrane dynamics, organelle division, and organelle biogenesis. Here, we summarize the major conceptual changes that have occurred in cell biology as we have come to appreciate how contact sites integrate the activities of organelles.

Form follows function: the importance of endoplasmic reticulum shape.

The endoplasmic reticulum (ER) has a remarkably complex structure, composed of a single bilayer that forms the nuclear envelope, along with a network of sheets and dynamic tubules. Our understanding of the biological significance of the complex architecture of the ER has improved dramatically in the last few years. The identification of proteins and forces required for maintaining ER shape, as well as more advanced imaging techniques, has allowed the relationship between ER shape and function to come into focus. These studies have also revealed unexpected new functions of the ER and novel ER domains regulating alterations in ER dynamics. The importance of ER structure has become evident as recent research has identified diseases linked to mutations in ER-shaping proteins. In this review, we discuss what is known about the maintenance of ER architecture, the relationship between ER structure and function, and diseases associated with defects in ER structure.

Uptake and trafficking of exogenous sterols in Saccharomyces cerevisiae.

The proper distribution of sterols among organelles is critical for numerous cellular functions. How sterols are sorted and moved among membranes remains poorly understood, but they are transported not only in vesicles but also by non-vesicular pathways. One of these pathways moves exogenous sterols from the plasma membrane to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae. We have found that two classes of proteins play critical roles in this transport, ABC transporters (ATP-binding-cassette transporters) and oxysterol-binding protein-related proteins. Transport is also regulated by phosphoinositides and the interactions of sterols with other lipids. Here, we summarize these findings and speculate on the role of non-vesicular sterol transfer in determining intracellular sterol distribution and membrane function.

Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae.

We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This receptor, along with SRP, targets ribosome-nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.

The protein translocation apparatus contributes to determining the topology of an integral membrane protein in Escherichia coli.

The assembly of integral membrane proteins is determined by features of these proteins and the protein translocation apparatus. We used alkaline phosphatase fusions to the membrane protein MalF to investigate the role of the protein translocation machinery in the arrangement of proteins in the cytoplasmic membrane of Escherichia coli. In particular, we studied the effects of prlA mutations on membrane protein topology. These mutations lie in the secY gene, which encodes a core component of the protein translocation apparatus. We find that the topology of some of the fusion proteins is changed and, in one case, is completely inverted in prlA mutants. We discuss the mechanism of prlA-mediated export and the role of the protein translocation apparatus in contributing to membrane protein topology.

The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm.

In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each other in vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.

Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm.

Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli.

Disulfide bonds are rarely found in cytoplasmic proteins. Mutations were selected for in Escherichia coli that allow disulfide bond formation in the cytoplasm. In the presence of these mutations, export-defective versions of alkaline phosphatase and mouse urokinase were able to fold into their enzymatically active conformations in the cytoplasm because their disulfide bonds were formed. The mutations were mapped to the gene for thioredoxin reductase and diminish or eliminate the activity of this enzyme. Thioredoxin itself was found to be unnecessary for this disulfide bond formation. Thioredoxin reductase, but not thioredoxin, is thus implicated in keeping cysteines reduced in cytoplasmic proteins.

Regional changes in adipose tissue blood flow and metabolism in rats after a meal.

Adipose tissue blood flow was measured in five depots, and plasma concentrations of glucose, insulin, and triglyceride were measured at 0, 15, 30, and 45 min after the start of a meal in unanesthetized, freely moving rats. In addition, adipose tissue lipoprotein lipase activity was measured in four depots before and 45 min after the start of a meal. Plasma glucose was significantly elevated only at the 15-min time point, and while plasma triglyceride increased these changes did not reach significance. Plasma insulin was significantly elevated at all time points after a meal. Feeding resulted in a consistent decrease of adipose tissue blood flow expressed per gram wet weight of tissue. This decrease was maximal at 30 min after the start of feeding. The decrease in adipose tissue blood flow averaged 45% at 45 min after the start of feeding for the five depots evaluated. Lipoprotein lipase activity significantly increased in the retroperitoneal and mesenteric fat depots at 45 min after the meal start, but did not change in the epididymal or dorsal subcutaneous fat depots. These results suggest that a decrease in adipose tissue blood flow is a normal result of a meal in the rat. The regional specificity of changes in adipose tissue lipoprotein lipase activity supports the concept of regional specificity of function for adipose tissue and suggests that the mesenteric and retroperitoneal depots are particularly important for the storage of triglycerides immediately after a meal.

Adipocyte blood flow is decreased in obese Zucker rats.

Blood flow to five adipose tissue depots and five other organs was measured in unanesthetized free-moving obese and lean male Zucker rats using the radiolabeled microsphere technique. Cardiac output and blood pressure were also measured. Obese rats were significantly heavier and had larger fat depots characterized by more and larger adipocytes. Cardiac output and blood pressure were similar in lean and obese rats. Blood flow to adipose tissue was substantially reduced in all five depots of obese rats when expressed per square millimeter of cell surface. When blood flow was expressed per cell, it was reduced in obese rats in the dorsal subcutaneous and inguinal depots and significantly elevated in the obese mesenteric adipose depot. Significant interdepot differences in blood flow were observed in both lean and obese rats. Nonadipose organ blood flow was not different between obese and lean rats, but when expressed on a per gram basis, blood flow through triceps surae muscle, epididymis, and testis was elevated in obese rats. These findings suggest that there is a substantial alteration of hemodynamics in obesity that may contribute to or reflect the altered metabolism of adipose tissue in obese rats. Furthermore, interdepot differences of adipose tissue blood flow also parallel reported interdepot differences in metabolism.

Human alpha-interferon enhances in vitro IgM rheumatoid factor synthesis by lymphocytes from normal subjects and rheumatoid arthritis patients.

Alpha-interferon (alpha-IFN) was added to pokeweed mitogen (PWM) or Epstein-Barr virus stimulated human peripheral blood mononuclear cell cultures from normal subjects or patients with rheumatoid arthritis (RA). Alpha-IFN enhanced in vitro production of PWM induced IgG and IgM, and significantly enhanced PWM induced IgM rheumatoid factor (IgM-RF) production by lymphocytes both from normal subjects and RA patients. Enhancement was recorded whether cells were preincubated with alpha-IFN for 16 hours or with alpha-IFN present throughout the culture period. Alpha-IFN did not enhance IgM-RF production in the absence of PWM or T cells. Enhancement of IgM-RF production was not seen in Epstein-Barr virus stimulated cultures.

alpha-Interferon increases immunoglobulin production in cultured human mononuclear leukocytes.

Interferons (IFN) are known to modulate immune responses in either an inhibitory or a stimulating manner. The present study was initiated to investigate the mechanisms by which alpha-IFN modulates Ig production of human peripheral blood mononuclear cells (PBMC). IgG and IgM production was measured in pokeweed mitogen- (PWM) stimulated 7-day cultures of PBMC. Significant enhancement of IgM and IgG production was observed when alpha-IFN was added. Overnight preincubation followed by washing also produced significant enhancement. The effect of alpha-IFN was not obtained in the absence of PWM or T cells. The effect of alpha-IFN on cultures of B and T cells was not altered by irradiation of T cells (2000 rad). alpha-IFN was not shown to enhance the production of helper factor but did increase the responsiveness of B cells to helper factor if the B cells were preincubated with alpha-IFN. Finally, alpha-IFN did not increase the Ig production of PBMC induced by Epstein Barr virus (EBV), and the outgrowth of EBV-infected PBMC was not affected. Overall, these results show for the first time that the effect of alpha-IFN on PBMC is due to an enhanced responsiveness of B cells to helper factors produced by radioresistant T cells.