Philipp E. Scherer

A Novel Serum Protein Similar to C1q, Produced Exclusively in Adipocytes

We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.

Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis

Caveolae, 50-100 nm invaginations that represent a subcompartment of the plasma membrane, have been known for many years, but their exact roles remain uncertain. The findings that the caveolae coat protein caveolin is a v-Src substrate and that G-protein-coupled receptors are present in caveolae have suggested a relationship between caveolae, caveolin and transmembrane signalling. The recent isolation of caveolin-rich membrane domains in which caveolin exists as a hetero-oligomeric complex with integral membrane proteins and known cytoplasmic signalling molecules provides support for this hypothesis. Compartmentalization of certain signalling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system.

Caveolae, transmembrane signalling and cellular transformation

Caveolae are approximately 50-100 nm membrane micro-invaginations associated with the plasma membrane of a wide variety of cells. Although they were first identified in transmission electron micrographs approximately 40 years ago, their exact function(s) has remained controversial. Two well-established functions include: (1) the transcytosis of both large and small molecules across capillary endothelial cells and (2) the utilization of GPI-linked proteins to concentrate small molecules in caveolae for translocation to the cytoplasm (termed potocytosis). Recently, interest in a third proposed caveolar function, namely transmembrane signalling, has been revived by the identification of caveolin--a transformation-dependent v-Src substrate and caveolar marker protein--and the isolation of caveolin-rich membrane domains from cultured cells. Here we will discuss existing evidence that suggests a role for caveolae in signalling events.

Caveolae purification and glycosylphosphatidylinositol-linked protein sorting in polarized epithelia.

Publisher Summary This chapter describes the techniques used for the recombinant expression of glycosylphosphatidylinositol (GPI)-linked proteins in epithelial cell lines and the measurement of cell-surface polarity of endogenous or transfected GPI-linked proteins at steady state and during transport. The chapter also discusses the methods for purification and characterization of caveolae from cultured cells. To study the sorting of endogenous GPI-linked proteins in polarized cells, a series of cell-surface labeling techniques that allow the rapid biochemical determination of the polarity of a given cell-surface antigen is developed. Such labeling techniques depend on the growth of polarized cells on permeable supports that allow for separate access to the apical and basolateral domains. These techniques are then applied to a variety of available intestinal and renal epithelial cell lines, such as the Madin-Darby canine kidney (MDCK), LLC-PK1, Caco-2, and SK-C015 lines, that spontaneously form polarized monolayers in culture. The GPI-linked proteins are detected by their sensitivity to release by treatment with bacterial PI-specific phospholipase C. To measure the polarized sorting of the recombinant proteins during cell-surface transport, additional assays are developed to monitor the cell surface delivery, endocytosis, and transcytosis.

Protein import into yeast mitochondria: The inner membrane import site protein ISP45 is the MPI1 gene product

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross‐linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p‐beta‐galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right‐side‐out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.

The primary sequence of murine caveolin reveals a conserved consensus site for phosphorylation by protein kinase C.

We report here the cloning of the murine cDNA encoding caveolin, a known v-Src substrate and caveolar marker protein. Interestingly, analysis of the murine cDNA and comparison with caveolin from other species reveals a previously unrecognized consensus site for protein kinase C (PKC) phosphorylation. This finding could have important implications as (i) both the morphology and function of caveolae are dramatically affected by PKC activators; and (ii) PKC alpha is concentrated in isolated caveolin-rich membrane domains. In addition, this first step should facilitate the use of the mouse as a genetic system for elucidating the role of caveolin in caveolar functioning.

Pantophysin is a phosphoprotein component of adipocyte transport vesicles and associates with GLUT4-containing vesicles.

Pantophysin, a protein related to the neuroendocrine-specific synaptophysin, recently has been identified in non-neuronal tissues. In the present study, Northern blots showed that pantophysin mRNA was abundant in adipose tissue and increased during adipogenesis of 3T3-L1 cells. Immunoblot analysis of subcellular fractions showed pantophysin present exclusively in membrane fractions and relatively evenly distributed in the plasma membrane and internal membrane fractions. Sucrose gradient ultracentrifugation demonstrated that pantophysin and GLUT4 exhibited overlapping distribution profiles. Furthermore, immunopurified GLUT4 vesicles contained pantophysin, and both GLUT4 and pantophysin were depleted from this vesicle population following treatment with insulin. Additionally, a subpopulation of immunopurified pantophysin vesicles contained insulin-responsive GLUT4. Consistent with the interaction of synaptophysin with vesicle-associated membrane protein 2 in neuroendocrine tissues, pantophysin associated with vesicle-associated membrane protein 2 in adipocytes. Furthermore, in [32P]orthophosphate-labeled cells, pantophysin was phosphorylated in the basal state. This phosphorylation was unchanged in response to insulin; however, insulin stimulated the phosphorylation of a 77-kDa protein associated with α-pantophysin immunoprecipitates. Although the functional role of pantophysin in vesicle trafficking is unclear, its presence on GLUT4 vesicles is consistent with the emerging role of solubleN-ethylmaleimide-sensitive protein receptor (SNARE) factor complex and related proteins in regulated vesicle transport in adipocytes. In addition, pantophysin may provide a marker for the analysis of other vesicles in adipocytes.

Diffuse vesicular distribution of Rab3D in the polarized neuroendocrine cell line AtT-20

The neuroendocrine cell line AtT‐20 has two types of storage vesicles: dense core granules and synaptic vesicles, both sequestered at the tip of the processes. Here we show that Rab3D protein, which is abundant in fat cells, is also expressed in AtT‐20 cells. Differently from Rab3A, which is localized in secretory vesicles accumulated at the tips, Rab3D has a diffuse vesicular distribution in the cytoplasm of the cell body, the processes and the tips. In AtT‐20 cells, Rab3D may define a regulated secretory pathway which functions independently from cell polarity.

The apical sorting of glycosylphosphatidylinositol-linked proteins

Publisher Summary Many cells, from yeast to human, use glycosylated form of phosphatidylinositol to anchor proteins to the cell surface. Several terms have been coined to describe this anchoring mechanism. They include “glycosylphosphatidylinositol (GPI) anchoring,” “glypiation,” “phosphatidylinositol-glycan (PIG) tailing,” and “greasy foot.” All GPI anchors contain a conserved glycan core structure, composed of ethanolamine, phosphate, mannose, glucosamine, and inositol, whereas GPI-linked proteins are found clustered in caveolae, a specialized domain of the plasma membrane. This chapter discusses the reasons for GPI-linked proteins being selectively transported to the apical surface of polarized epithelial cells, with GPI acting as a dominant apical trafficking signal.