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L J Urbani

Publications and source records attributed to L J Urbani.

6 recordsLinked to original sources
PubMed1984

Transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system.

Mixed monolayers containing vesicular stomatitis virus-infected Chinese hamster ovary clone 15B cells (lacking UDP-N-acetylglucosamine transferase I, a Golgi enzyme) and uninfected wild-type Chinese hamster ovary cells were formed. Extensive cell fusion occurs after the monolayer is exposed to a pH of 5.0. The vesicular stomatitis virus encoded membrane glycoprotein (G protein) resident in the rough endoplasmic reticulum (labeled with [35S]methionine) or Golgi complex (labeled with [3H]palmitate) of 15B cells at the time of fusion can reach Golgi complexes from wild-type cells after fusion; G protein present in the plasma membrane cannot. Transfer to wild-type Golgi complexes is monitored by the conversion of G protein to an endoglycosidase H-resistant form upon arrival, and also demonstrated by immunofluorescence microscopy. G protein in the Golgi complex of the 15B cells at the time of fusion exhibits properties vis a vis its transfer to an exogenous Golgi population identical to those found earlier in a cell-free system (Fries, E., and J. E. Rothman. 1981. J. Cell Biol., 90: 697-704). Specifically, pulse-chase experiments using the in vivo fusion and in vitro assays reveal the same two populations of G protein in the Golgi complex. The first population, consisting of G protein molecules that have just received their fatty acid, can transfer to a second Golgi population in vivo and in vitro. The second population, entered by G protein approximately 5 min after its acylation, is unavailable for this transfer, in vivo and in vitro. Presumably, this second population consists of those G-protein molecules that had already been transferred between compartments within the 15B Golgi population, in an equivalent process before cell fusion or homogenization for in vitro assays. Evidently, the same compartment boundary in the Golgi complex is detected by these two measurements. The surprisingly facile process of glycoprotein transit between Golgi stacks that occurs in vivo may therefore be retained in vitro, providing a basis for the cell-free system.

Acetylglucosamine↗
PubMed1984

Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations.

The transfer of the vesicular stomatitis virus-encoded glycoprotein (G protein) between Golgi populations in fused cells (Rothman, J. E., L. J. Urbani, and R. Brands. 1984. J. Cell Biol. 99:248-259) is exploited here to study and to help define the compartmental organization of the Golgi stack and to characterize the mechanism of intercompartmental transport. We find that G protein that has just received its peripheral N-acetylglucosamine in the Golgi complex of one cell is efficiently transferred to the Golgi complex of another cell to receive galactose (Gal). Remarkably, this transport occurs at the same rate between these two compartments whether they are present in the same or different Golgi populations. Therefore, a dissociative (presumably vesicular) transport step moves G protein from one part of the Golgi in which N-acetylglucosamine is added to another in which Gal is added. Minutes later, upon receiving Gal, the same G protein molecules are very poorly transferred to an exogenous Golgi population after cell fusion. Therefore, once this intercompartmental transfer has already taken place (before fusion), it cannot take place again (after fusion); i.e., transport across the compartment boundary in the Golgi complex that separates the sites of N-acetylglucosamine and Gal incorporation is a vectorial process. We conclude that transfers between Golgi cisternae occur by a stochastic process in which transport vesicles budding from cisternae dissociate, can diffuse away, and then attach to and fuse with the appropriate target cisterna residing in the same or in a different stack, based on a biochemical pairing after a random encounter. Under these circumstances, a transported protein would almost always randomize among stacks with each intercisternal transfer; it would not progress systematically through a single stack. Altogether, our studies define three sequential compartments in the Golgi stack.

Acetylglucosamine↗
PubMed1981

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Enzymes that catalyze the two successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary cells were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, alpha-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitis viral G protein (to yield a "trimmed" oligosaccharide core) was separated from enzymes (galactosyl- and sialyltransferases) that act in the later, terminal stage of glycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase, whereas G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of two distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. It is likely that these compartments consist of cisternae on the cis and trans sides of the Golgi stack.

Animals↗
PubMed1980

Relationship of antibody to outcome in neonatal herpes simplex virus infections.

Neutralizing antibody titers to herpes simplex virus type 1 (HSV-1) and HSV-2 were measured at birth in normal infants and uninfected infants of mothers with genital HSV infections during pregnancy and at the onset of infection in 5 infants with mild infections and 11 infants with severe infections. Thirty-eight percent of premature and 29% of term infants had neutralization titers of <1:5. High titers ([unk]1:40) were found in 55% of infants of mothers with primary infections during pregnancy and in 76% of infants of mothers with recurrent infections. The mean titers to HSV-1 and -2 in 5 infected infants with mild infections were 1:56 and 1:65 at the time of onset of infection, whereas the mean titers in 11 infants with severe infections were 1:11 and 1:12. Six natally exposed infants who remained asymptomatic were also studied and had a mean titer to HSV-1 of 1:85 and to HSV-2 of 1:69. Therefore, infants with high titers of transplacentally derived antibody had a more favorable outcome than infants with lower titers. Ninety-five percent of the infants of mothers with recurrent infections had a Rawls index of more than 85, suggesting that the antibody response was to HSV-2. However, low levels of antibody with this type specificity failed to protect four infants from infection with HSV-2. Augmentation of the neutralization titer to HSV-2 by the amount of complement present in cord serum was less than twofold. The study suggests that the quantity of antibody derived transplacentally affects the outcome of infection after natal exposure to herpes simplex virus. Complete neutralization of virus by antibody may occur in some infants, and prolongation of the incubation period and modification of the infection may occur in others.

Age Factors↗