Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such “membrane-inserted” form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKCα, the C2–V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes.

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol. Chem. 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed "caves," using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria

Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles.

Copper exposure induces trafficking of the Menkes protein in intestinal epithelium of ATP7A transgenic mice

The final steps in the absorption and excretion of copper at the molecular level are accomplished by 2 closely related proteins that catalyze the ATP-dependent transport of copper across the plasma membrane. These proteins, ATP7A and ATP7B, are encoded by the genes affected in human genetic copper-transport disorders, namely, Menkes and Wilson diseases. We studied the effect of copper perfusion of an isolated segment of the jejunum of ATP7A transgenic mice on the intracellular distribution of ATP7A by immunofluorescence of frozen sections. Our results indicate that ATP7A is retained in the trans-Golgi network under copper-limiting conditions, but relocalized to a vesicular compartment adjacent to the basolateral membrane in intestines perfused with copper. The findings support the hypothesis that the basolateral transport of copper from the enterocyte into the portal blood may involve ATP7A pumping copper into a vesicular compartment followed by exocytosis to release the copper, rather than direct pumping of copper across the basolateral membrane.

The role of copper transport proteins in the small intestines

Physiological copper homeostasis involves striking a balance between absorption and secretion. ATP7A was identified at the trans-Golgi network but relocalized to vesicles under copper exposure in the intestine. This suggests that ATP7A may be a rate limiting step in intestinal uptake of copper.

Regulation of HbPIP2;3, a latex-abundant water transporter, is associated with latex dilution and yield in the rubber tree (Hevea brasiliensis Muell. Arg.)

Rubber tree (Hevea brasiliensis) latex, the source of natural rubber, is synthesised in the cytoplasm of laticifers. Efficient water inflow into laticifers is crucial for latex flow and production since it is the determinant of the total solid content of latex and its fluidity after tapping. As the mature laticifer vessel rings are devoid of plasmodesmata, water exchange between laticifers and surrounding cells is believed to be governed by plasma membrane intrinsic proteins (PIPs). To identify the most important PIP aquaporin in the water balance of laticifers, the transcriptional profiles of ten-latex-expressed PIPs were analysed. One of the most abundant transcripts, designated HbPIP2;3, was characterised in this study. When tested in Xenopus laevis oocytes HbPIP2;3 showed a high efficiency in increasing plasmalemma water conductance. Expression analysis indicated that the HbPIP2;3 gene was preferentially expressed in latex, and the transcripts were up-regulated by both wounding and exogenously applied Ethrel (a commonly-used ethylene releaser). Although regular tapping up-regulated the expression of HbPIP2;3 during the first few tappings of the virginal rubber trees, the transcriptional kinetics of HbPIP2;3 to Ethrel stimulation in the regularly tapped tree exhibited a similar pattern to that of the previously reported HbPIP2;1 in the virginal rubber trees. Furthermore, the mRNA level of HbPIP2;3 was associated with clonal yield potential and the Ethrel stimulation response. Together, these results have revealed the central regulatory role of HbPIP2;3 in laticifer water balance and ethylene stimulation of latex production in Hevea.

Endosomal trafficking of the Menkes copper ATPase ATP7A is mediated by vesicles containing the Rab7 and Rab5 GTPase proteins

The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 μM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.

A short-term, high-fat diet up-regulates lipid metabolism and gene expression in human skeletal muscle

Background: Dietary fatty acids may be important in regulating gene expression. However, little is known about the effect of changes in dietary fatty acids on gene regulation in human skeletal muscle.
Objective: The objective was to determine the effect of altered dietary fat intake on the expression of genes encoding proteins necessary for fatty acid transport and ß-oxidation in skeletal muscle.
Design: Fourteen well-trained male cyclists and triathletes with a mean (± SE) age of 26.9 ± 1.7 y, weight of 73.7 ± 1.7 kg, and peak oxygen uptake of 67.0 ± 1.3 mL ˙ kg-1 ˙ min-1 consumed either a high-fat diet (HFat: > 65% of energy as lipids) or an isoenergetic high-carbohydrate diet (HCho: 70–75% of energy as carbohydrate) for 5 d in a crossover design. On day 1 (baseline) and again after 5 d of dietary intervention, resting muscle and blood samples were taken. Muscle samples were analyzed for gene expression [fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), carnitine palmitoyltransferase I (CPT I), ß-hydroxyacyl-CoA dehydrogenase (ß-HAD), and uncoupling protein 3 (UCP3)] and concentrations of the proteins FAT/CD36 and FABPpm.
Results: The gene expression of FAT/CD36 and &szlig; -HAD and the gene abundance of FAT/CD36 were greater after the HFat than after the HCho diet (P < 0.05). Messenger RNA expression of FABPpm, CPT I, and UCP-3 did not change significantly with either diet.
Conclusions: A rapid and marked capacity for changes in dietary fatty acid availability to modulate the expression of mRNA-encoding proteins is necessary for fatty acid transport and oxidative metabolism. This finding is evidence of nutrient-gene interactions in human skeletal muscle.

Recent advances in chloroplast and mitochondrial division

Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely expressed in various tissues. Orthologues are found in vertebrates, invertebrates, plants, and microorganisms. Related proteins exhibit a common core structure, termed the prohibitin (PHB) domain, with varying extensions. Stomatin has an unusual topology, similar to caveolin-1, with a hydrophobic domain embedded at the cytoplasmic side of the membrane. Additional anchoring is provided by palmitoylation and the membrane affinity of the PHB domain. Stomatin associates with cholesterol-rich microdomains (lipid rafts), forms oligomers, and thereby displays a scaffolding function by generating large protein-lipid complexes. It regulates the activity of various membrane proteins by reversibly recruiting them to lipid rafts. This mechanism of regulation has been shown for GLUT-1 and may also apply for ion channels. Stomatin is located at the plasma membrane, particularly in microvilli, in endocytic and exocytic vesicles, and cytoplasmic granules. Stomatin-carrying endosomes are highly dynamic and interact with lipid droplets suggesting a role in intracellular lipid transport. This subcellular distribution and the caveolin-like protein structure suggest important membrane organizing functions for stomatin. A general picture emerges now that cell membranes contain cholesterol-rich domains that are generated and regulated by scaffolding proteins like caveolins, stomatins, and flotillin/reggie proteins.

Creatine transporters: a reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na⁺/Cl^--dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.
In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the agr-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.
Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions.
In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with ¹⁴C-Cr-isotope tracer studies and a number of [³¹P]-NMR magnetization transfer studies, as well as with recent [¹H]-NMR spectroscopy data.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells.

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 °C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.