A new model for nuclear envelope breakdown.

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

A visual screen of a GFP-fusion library identifies a new type of nuclear envelope membrane protein.

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

Binding of signal recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction.

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC-SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

Cholesterol-independent targeting of Golgi membrane proteins in insect cells.

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.

Identification of a cellular receptor for subgroup E avian leukosis virus.

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.

CAR1, a TNFR-related protein, is a cellular receptor for cytopathic avian leukosis-sarcoma viruses and mediates apoptosis.

Viral envelope (Env)-receptor interactions have been implicated in the cell death associated with infection by subgroups B and D avian leukosis-sarcoma viruses (ALVs). A chicken protein, CAR1, was identified that permitted infection of mammalian cells by these viral subgroups. CAR1 bound to a viral Env fusion protein, comprising an ALV-B surface Env protein and the Fc region of an immunoglobulin, indicating that it is a specific viral receptor. CAR1 contains two extracellular cysteine-rich domains characteristic of the TNFR family and a cytoplasmic region strikingly similar to the death domain of TNFR1 and Fas, implicating this receptor in cell killing. Chicken embryo fibroblasts susceptible to ALV-B infection and transfected quail QT6 cells expressing CAR1 underwent apoptosis in response to the Env-Ig fusion protein, demonstrating that this cytopathic ALV receptor can mediate cell death.

Approaching the mechanism of protein transport across the ER membrane.

Since the identification of essential protein-translocation components in the endoplasmic reticulum membrane, research efforts have concentrated on the elucidation of the molecular mechanism of protein transport across this membrane. Recent results have provided new information as to how proteins are targeted to, and inserted into, the translocation site during translation. Post-translational translocation has also been examined and is distinct from cotranslational translocation with respect to the mechanism and membrane protein components involved.

Expression of additional genes in a vector derived from a minimal RNA virus.

Previous studies have shown that expression of the vesicular stomatitis virus (VSV) glycoprotein (G) from a Semliki Forest virus (SFV) RNA replicon results in the production of propagating infectious particles that we call minimal viruses. These minimal viruses consist of vesicles containing VSV G protein that bud from the plasma membrane and trap the infectious SFV G RNA, but they do not contain other viral structural proteins. The cell binding and membrane fusion activity of the VSV G protein allow minimal viruses to propagate in tissue culture cells. To determine if these minimal viruses could be used to express foreign genes, we added a second SFV promoter and a multiple cloning site downstream of the VSV G gene. We report here expression of three different proteins from this modified, minimal virus vector. Although expression of each foreign, unselected gene was lost rapidly from the vector upon passaging, it was possible after the initial transfection to derive stocks of infectious particles that could be used to infect multiple additional cultures and transfer protein expression efficiently. When cells were infected with these minimal viruses, host protein synthesis was shut off and the foreign protein and VSV G proteins were the major proteins expressed in the infected cells. Both were expressed at similar levels and accumulated to about 1-2% of total cell protein.

Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA.

Self-propagating infectious particles were produced in animal cells transfected with an RNA replicon encoding a single viral structural protein, the vesicular stomatitis virus glycoprotein (VSV-G). The replicon is derived from an alphavirus, Semliki Forest virus (SFV), and encodes the SFV RNA replicase, but none of the SFV structural proteins. After transfection of the replicon into tissue culture cells, expression of G protein spread from small foci throughout the culture. Supernatants from the cells contained infectious, virus-like particles that could be passaged and were neutralized by anti-VSV serum. The majority of the infectious particles were smaller and less dense than either VSV or SFV. Characterization by electron microscopy showed membrane-enveloped vesicles that contained the VSV-G protein. Infectious particles were apparently generated by budding of vesicles containing VSV-G protein and the RNA replicon. These experiments reveal that an enveloped infectious agent can be much simpler than previously thought.

Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.