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Biochemical studies on rat liver Golgi apparatus. III. Subfractionation of fragmented Golgi apparatus by counter-current distribution.

Vesicular fragments of Golgi apparatus, smooth- and rough-surfaced microsomes from rat liver are differently partitioned in aqueous polymer two-phase systems consisting of dextran, polyethylene glycol, and sodium phosphate buffer. At a given polymer concentration, the amount of material partitioned in the top phase increases in the following order: rough microsomes less than smooth microsomes less than Golgi fragments. Counter-current distribution of Golgi fragments in the system consisting of 6.8% (w/w) dextran T500 and 6.8% polyethylene glycol 4,000 results in the separation of the fragments into three fractions; i.e. Fractions I, II, and III. NADH- and NADPH-cytochrome c reductase activities are detected almost exclusively in Fraction I, whereas the activities of galactosyltransferase, acid phosphatase, 5'-nucleotidase, and thiamine pyrophosphatase are maximal in Fraction III and minimal in Fraction I. The distribution of these enzymes suggests that Fraction I is similar to, though not identical with, microsomes, Fraction III resembles plasma membrane and lysosomes, and Fraction II is between the two. It is concluded that NADH- and NADPH-cytochrome c reductases are localized in a restricted region of the Golgi structure and that intra-Golgi differentiation seems to proceed in a discontinuous manner.

Acid Phosphatase↗

IcsA, a polarly localized autotransporter with an atypical signal peptide, uses the Sec apparatus for secretion, although the Sec apparatus is circumferentially distributed.

Asymmetric localization of proteins is essential to many biological functions of bacteria. Shigella IcsA, an outer membrane protein, is localized to the old pole of the bacillus, where it mediates assembly of a polarized actin tail during infection of mammalian cells. Actin tail assembly provides the propulsive force for intracellular movement and intercellular dissemination. Localization of IcsA to the pole is independent of the amino-terminal signal peptide (Charles, M., Perez, M., Kobil, J.H., and Goldberg, M.B., 2001, Proc Natl Acad Sci USA 98: 9871-9876) suggesting that IcsA targeting occurs in the bacterial cytoplasm and that its secretion across the cytoplasmic membrane occurs only at the pole. Here, we characterize the mechanism by which IcsA is secreted across the cytoplasmic membrane. We present evidence that IcsA requires the SecA ATPase and the SecYEG membrane channel (translocon) for secretion. Our data suggest that YidC is not required for IcsA secretion. Furthermore, we show that polar localization of IcsA is independent of SecA. Finally, we demonstrate that while IcsA requires the SecYEG translocon for secretion, components of this apparatus are uniformly distributed within the membrane. Based on these data, we propose a model for coordinate polar targeting and secretion of IcsA at the bacterial pole.

Adenosine Triphosphatases↗

Suspensory apparatus prosthesis in the horse. Part 2: In vivo evaluation in a suspensory apparatus disruption model.

The suspensory apparatus of one forelimb was surgically disrupted in six adult horses by transecting the distal sesamoidean ligaments. A double-braided prosthetic ligament made of aramid yarn was installed to support the flexor surface of the metacarpophalangeal joint. The prosthesis was routed through tunnels in the third metacarpal bone and proximal phalanx, and secured to bone with screws. Evaluation by radiography, synovial fluid analysis, cinematography, and dynamography was performed before surgery and at weeks 16 and 30. Supracondylar cortical lysis and periosteal proliferation were observed on postoperative radiographs. Synovitis and fragmentation of the prosthesis were apparent from synovial fluid evaluation. Weight bearing and metacarpophalangeal joint motion were decreased and loading was transferred in part to the opposite forelimb. Clinical lameness improved and weight bearing increased during the second half of the 30-week period. At necropsy, there was abrasion of the prosthesis and the articular surfaces in contact with the prosthesis. Diffuse granulomatous synovitis developed in response to aramid fiber fragments within the synovium.

Animals↗