Marilyn G. Farquhar

Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum.

Newly synthesized proteins are believed to move from the endoplasmic reticulum (ER) to the Golgi by bulk flow, and sorting is assumed to occur exclusively in the trans-Golgi network (TGN). Using quantitative immunoelectron microscopy, we demonstrate that vesicular stomatitis virus glycoprotein (VSV-G) is sorted from resident ER proteins and concentrated 5- to 10-fold in 40-80 nm vesicles during vesicle budding from the ER. Accumulation of VSV-G in pre-Golgi vesicular carriers is the only detectable concentration step in its transport to the TGN. From these results, it is apparent that export from the ER is not exclusively mediated by bulk flow. The ER exerts an unanticipated level of control to insure selective and efficient entry of mature protein into the secretory pathway.

The Golgi apparatus: 100 years of progress and controversy

Research on the Golgi apparatus has resulted in major advances in understanding its structure and functions, but many important questions remain unanswered. The history of the Golgi apparatus has been marked by arguments and controversies, some of which have been resolved, whereas others are still ongoing. This article charts progress in understanding the role of the Golgi apparatus during the 100 years since it was discovered, highlighting major milestones and discoveries that have led to the concepts of the organization and functions of this organelle that we have today.

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na(+)/H(+)-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside-, protamine sulfate-, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium.

The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae

Mannose-6-phosphate (Man-6-P) receptors for lysosomal enzymes were localized by immunocytochemistry in several secretory and adsorptive cell types using monospecific antireceptor antibodies. By immunofluorescence, the receptors were found in the Golgi region of polarized cells. When localized by immunoperoxidase at the electron microscope level, they were detected in Golgi cisternae, coated vesicles, endosomes, and lysosomes of all cell types examined (hepatocytes, exocrine pancreatic and epididymal epithelia). Within the Golgi complex, immunoreactive receptors were restricted in their distribution to one or two cisternae on the cis side of the Golgi stacks. They were not detected in trans Golgi or GERL cisternae. Based on their high concentration of Man-6-P receptors, we propose that the cis Golgi cisternae represent the site where the secretory and lysosomal pathways diverge: lysosomal enzymes bearing the Man-6-P recognition marker bind to Man-6-P receptors in this location and are delivered to endosomes and lysosomes via coated vesicles.

GOLPH3 Bridges Phosphatidylinositol-4- Phosphate and Actomyosin to Stretch and Shape the Golgi to Promote Budding

Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid-binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends on PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, β1-adrenergic receptors (β1-AR), β2-AR, Gαs, and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (β-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and α-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/μm of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Gαs from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-Gs-AC in lipid rafts/caveolae.

RGS proteins: more than just GAPs for heterotrimeric G proteins

Abstract Members of the newly described RGS family of proteins have a common RGS domain that contains GTPase-activating activity for many Gα subunits of heterotrimeric G proteins. Their ability to dampen signalling via Gα i -, Gα q - and Gα 12/13 -coupled pathways makes them crucial players in mediating the multitude of cellular processes controlled by heterotrimeric G proteins. Some RGS proteins also contain additional motifs that link them to other signalling networks, where they constitute effector-type molecules. This review summarizes recent findings on RGS proteins, especially those that implicate RGS proteins in more than just enhancing the GTPase activity of their Gα subunit targets.

The formation of golgi stacks from vesiculated golgi membranes requires two distinct fusion events

We have reconstituted the fusion and assembly of vesiculated Golgi membranes (VGMs) into functionally active stacks of cisternae. A kinetic analysis of this assembly process revealed that highly dispersed VGMs of 60-90 nm diameter first fuse to form larger vesicles of 200-300 nm diameter that are clustered together. These vesicles then fuse to form tubular elements and short cisternae, which finally assemble into stacks of cisternae. We now provide evidence that the sequential stack formation from VGMs reflects two distinct fusion processes: the first event is N-ethyl-maleimide (NEM)-sensitive factor (NSF) dependent, and the second fusion event requires an NSF-like NEM-sensitive ATPase called p97. Interestingly, while the earliest steps in stack formation share some similarities with events catalyzing fusion of transport vesicles to its target membrane, neither GTP gamma S nor Rab-GDI, inhibitors of vesicular protein traffic, inhibit stack formation.

Comparative distribution of laminin, type IV collagen, and fibronectin in the rat glomerulus.

The distribution of laminin, type IV collagen, and fibronectin was investigated in the rat kidney cortex by immunolabeling. It was demonstrated by immunofluorescence on both unfixed cryostat sections and fixed ultracryomicrotome sections, by immunoperoxidase on fixed cryostat sections, and by immunoferritin on isolated glomerular basement membranes (GBM). This multifaceted approach provided complementary and convergent results. Distinct patterns were found for each antigen in the glomerulus and remaining kidney cortex. Laminin was localized predominantly in the GBM, where it was concentrated in the laminae rarae. Staining also occurred to a lesser extent in the mesangial matrix. Type IV collagen was evenly distributed in the lamina densa of the GBM and in the mesangial matrix. Fibronectin was most abundant in the mesangial matrix, but it could also be detected in the peripheral GBM, where it was localized in the laminae rarae. Labeling for fibronectin was particularly prominent at the endothelial-mesangial interface. The findings indicate that the three layers of the GBM differ in their composition: The lamina densa contains type IV collagen and the laminae rarae contain the two attachment proteins, fibronectin and laminin. The mesangial matrix stains for all three antigens, but it is also heterogeneous and can be subdivided into several domains--i.e., the endothelial-mesangial matrix, which is particularly rich in fibronectin, the intermesangial matrix, which contains mainly type IV collagen and fibronectin, and the GBM (where it continues over the mesangial regions), which stains most heavily for laminin.

DIVERGENCE OF RGS PROTEINS: EVIDENCE FOR THE EXISTENCE OF SIX MAMMALIAN RGS SUBFAMILIES

The authors thank Russell Doolittle for reading the manuscript. This work was supported by National Institutes of Health Grants DK 17780 and CA58689 (to M. G. F.). B. Z. is a member of the Molecular Pathology Graduate Program, University of California San Diego, and is supported by the HUANG Memorial Scholarship.We would like to apologize to investigators whose work could not be cited here due to space limitations.