Translocational pausing of apolipoprotein B can be regulated by membrane lipid composition.

One potential mechanism by which apolipoprotein (apo) B secretion is regulated is via transient pausing during translocation across the endoplasmic reticulum membrane. We have previously shown that translocation and secretion of full-length and truncated variants of apoB 100 are impaired in hepatocytes in which microsomal membranes are enriched in the phospholipid phosphatidylmonomethylethanolamine (PMME). We have now investigated whether or not the decreased translocation of apoB is the result of altered membrane lipid composition having an impact on translocational pausing. Our experiments showed that less in vitro translated apoB-15 (the N-terminal 15% of human apoB-100) was translocated into the lumen of PMME-enriched microsomes than of control microsomes. Proteinase K treatment of the translocation products yielded discrete N-terminal fragments of apoB indicating that both types of microsomal membranes contained translocationally paused nascent chains. Similarly, apoB generated from a truncated mRNA lacking a stop codon was also found to be translocationally paused. However, restarting of translocation after translocational pausing was impaired in PMME-enriched, but not in control, microsomes. These data suggest that secretion of apoB-containing lipoproteins can be regulated by membrane lipid composition at the level of translocational pausing.

Calnexin and other factors that alter translocation affect the rapid binding of ubiquitin to apoB in the Sec61 complex.

Several secretory proteins, including apolipoprotein B, have been shown to undergo degradation by proteasomes. We found that the rapid degradation of nascent apolipoprotein B in HepG2 cells was diminished but not abolished by the addition of any of three different inhibitors of proteasomes. Ubiquitin is conjugated to apolipoprotein B that is not assembled with sufficient lipids either during or soon after synthesis. In addition, we found that apolipoprotein B that has entered the endoplasmic reticulum sufficiently to become glycosylated can be degraded by proteasomes. Furthermore, we detected ubiquitin-apolipoprotein B that is associated with the Sec61 complex, the major constituent of the translocational channel. Treatment of cells with monomethylethanolamine or dithiothreitol decreased the translocation of apolipoprotein B and increased the proportion of ubiquitin-conjugated molecules associated with Sec61. Conversely, treatment of cells with oleic acid, which increased the proportion of translocated apolipoprotein B, decreased the amount of ubiquitin-apolipoprotein B in the Sec61 complex. Finally, we found that inhibition of the interaction between calnexin and apolipoprotein B decreases the translocation of apolipoprotein B, increases the ubiquitin-apolipoprotein B in the Sec61 complex, and increases the proteasomal degradation of glycosylated apolipoprotein B. Thus, ubiquitin can be attached to unassembled apolipoprotein B in the Sec61 complex, and this process is affected by factors including calnexin that alter the translocation of apolipoprotein B.

Translocational pausing is a common step in the biogenesis of unconventional integral membrane and secretory proteins.

Signal, stop transfer, and signal-anchor sequences direct a nascent polypeptide to a single topology with respect to the membrane of the endoplasmic reticulum. However, other types of sequences direct nascent proteins, either transiently or permanently, to more than one topologic form. Pause transfer sequences direct nascent apolipoprotein B to pause during its translocation, resulting in nonintegrated, transmembrane intermediates that become fully translocated over time. The stop transfer effector sequence (STE) directs the nascent prion protein either to integrate at the hydrophobic domain which immediately follows (TM1) or to become fully translocated, in a manner dependent on cytosolic factors. Although the action of pause transfer sequences has been dissected into stop and restart steps, the mechanism of STE action is unknown. Using chimeric proteins expressed in vitro, we show that STE, independent of TM1, acts as a pause transfer sequence. We also demonstrate that translocational pausing at STE is a common step preceding either complete translocation or integration into the membrane of a chimeric protein containing STE and TM1. These findings have implications for the role of pausing in the biogenesis of both secretory and membrane proteins.

Analysis of a pause transfer sequence from apolipoprotein B.

In contrast to typical secretory proteins, apolipoprotein B pauses at distinct points along the nascent chain during its translocation into the lumen of the endoplasmic reticulum. Specific pause transfer sequences mediate such discrete pauses in the translocation of apolipoprotein B. These sequences have been shown to confer this translocational behavior to heterologous chimeric proteins. To investigate the function of pause transfer sequences, we: (i) examine whether the multiple pause transfer sequences of apolipoprotein B act independently or are dependent upon the action of upstream pause transfer sequences, (ii) identify residues of the prototypical B' pause transfer sequence that are involved in pausing, and (iii) determine whether the stopping step of a translocational pause is a consequence of translational pausing, as has been suggested by other investigators. We conclude that pause transfer sequences act independently of each other and of translation; translocational pausing occurs even in the absence of ongoing protein synthesis. Furthermore, like other topogenic sequences such as signal sequences, pause transfer sequences are degenerate in structure yet have distinctive features necessary for their action. This characterization of the B' pause transfer sequence may aid in the identification of such sequences elsewhere in apolipoprotein B and in other proteins and has implications for the mechanism of translocational pausing.

Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation.

Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo B15) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo B15 appears to stop and restart translocation at several points. We have identified a sequence of amino acids in apo B15 that confers this behavior on a heterologous chimeric protein. In addition, we dissect pausing into two distinct steps, stopping and restarting, thereby trapping otherwise transient intermediates. Finally, we demonstrate the function of a second "pause transfer" sequence over 200 amino acids downstream in apo B15 that restarts translocation posttranslationally, suggesting that multiple pause transfer sequences are involved in the biogenesis of apolipoprotein B.

New variation on the translocation of proteins during early biogenesis of apolipoprotein B.

Apolipoprotein B (apo B) is crucial for the transport of cholesterol in humans. It is a large secretory protein that mediates the uptake of low-density lipoproteins and renders several forms of lipid droplets soluble in the blood. The binding of lipid by apo B also prevents this hydrophobic protein from precipitating in aqueous solution. In the endoplasmic reticulum, nascent secretory proteins must be translocated through an aqueous channel in the membrane into the aqueous lumen, so some novel form of processing may be necessary to maintain the solubility of apo B during its translocation. We have discovered that the biogenesis of apo B in cell-free systems does indeed involve a new variation on protein translocation: unlike typical secretory proteins, apo B is synthesized as a series of transmembrane chains with large cytoplasmic domains and progressively longer amino-terminal regions that are protected against added proteases during the translocation process. In contrast to typical transmembrane proteins, these transmembrane chains are not integrated into the bilayer. Moreover, the transmembrane chains with the shortest protected domains are precursors of forms whose protection is progressively extended to cover the length of the protein. This stepwise conversion occurs post-translationally for the most part. We propose a model on the basis of these findings for the biogenesis of apo B.

Infections with Cryptococcus neoformans in the acquired immunodeficiency syndrome.

We reviewed the records of 106 patients with cryptococcal infections and the acquired immunodeficiency syndrome (AIDS) treated at San Francisco General Hospital. We examined four issues: the efficacy of treatment with amphotericin plus flucytosine as compared with amphotericin alone, the efficacy of suppressive therapy, the prognostic clinical characteristics, and the course of nonmeningeal cryptococcosis. In 48 of the 106 patients (45 percent), cryptococcosis was the first manifestation of AIDS. Among the 89 patients with cryptococcal meningitis confirmed by culture, survival did not differ significantly between those treated with amphotericin plus flucytosine (n = 49) and those treated with amphotericin alone (n = 40). Flucytosine had to be discontinued in over half the patients because of cytopenia. Long-term suppressive therapy with either ketoconazole or amphotericin was associated with improved survival, as compared with survival in the absence of suppressive therapy (median survival, greater than or equal to 238 vs. 141 days; P less than 0.004). The only clinical features independently associated with a shorter cumulative survival were hyponatremia and a positive culture for cryptococcus from an extrameningeal source. The 14 patients with nonmeningeal cryptococcosis had a median survival (187 days) and rate of relapse (20 percent) similar to those in the patients with meningitis (165 days and 17 percent, respectively). From this retrospective study of cryptococcal infections in patients with AIDS we conclude that the addition of flucytosine to amphotericin neither enhances survival nor prevents relapse, but long-term suppressive therapy appears to benefit these patients.