Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism

In an in vitro system comprising a yeast cell-free translation system, yeast microsomes and mRNA encoding prepro-alpha-factor, the translocation of this protein across the membrane of the microsomal vesicle and its glycosylation could b uncoupled from its translation. Such post-translational processing is dependent upon the presence of ATP in the system. It is not, however, affected by a variety of uncouplers, ionophores or inhibitors, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), valinomycin, nigericin, dinitrophenol (DNP), potassium cyanide (KCN) or N-ethyl maleimide (NEM). This mechanism of translocation is significant as it indicates that a protein of 18 000 daltons is capable of crossing an endoplasmic reticulum-derived membrane post-translationally. For the moment, this phenomenon seems to be restricted to prepro-alpha-factor in the yeast in vitro system. Neither invertase nor IgG chi light chain could be translocated post-translationally in yeast, nor was such processing observed for prepro-alpha-factor in a wheat germ system supplemented with canine pancreatic microsomes.     

Secretion in yeast. Purification and in vitro translocation of chemical amounts of prepro-alpha-factor

The Saccharomyces cerevisiae mating pheromone precursor, prepro-alpha-factor, can be translocated across yeast endoplasmic reticulum membranes post-translationally in an in vitro system. This characteristic makes prepro-alpha-factor potentially useful as a probe in the biochemical dissection of the mechanism of this basic cellular process. Efforts have been limited by the inability to isolate sufficient quantities of such secretory protein precursors in a translocation-competent form. We report here the one-step purification of chemical amounts of translocation-competent prepro-alpha-factor using nickel ion affinity chromatography on nitrilotriacetate resin. An oligonucleotide encoding 6 histidine residues was inserted into a genomic clone encoding prepro-alpha-factor 5' of the naturally occurring translational stop codon by site-directed mutagenesis. The construct was expressed at high levels in a SecY- strain of Escherichia coli. The produced preprotein was solubilized in 6 M guanidine hydrochloride and bound to nitrilotriacetate resin. Prepro-alpha-factor was recovered at a purity in excess of 95% by elution with 0.25 M imidazole, 8 M urea, which competitively displaced the histidine affinity tag from the nickel column. The chemical amounts of prepro-alpha-factor obtained in this way were determined to be competent for translocation across yeast microsomal membranes and for subsequent modifications such as signal sequence cleavage and N-linked glycosylation.     

In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor

The in vitro synthesized precursor of the alpha-factor pheromone, prepro-alpha-factor, of Saccharomyces cerevisiae was translocated across yeast microsomal membranes in either a homologous or a wheat germ cell free system. Translocated prepro-alpha-factor was glycosylated, sedimented with yeast microsomal vesicles, and was protected from digestion by added protease, but was soluble after alkaline sodium carbonate treatment. Thus prepro-alpha-factor was properly sequestered within yeast microsomal vesicles, but was not integrated into the lipid bilayer. In marked contrast to protein translocation across mammalian microsomal membranes, translocation of prepro-alpha-factor across yeast microsomal membranes could occur posttranslationally. This reaction required protein components in the yeast microsomal fraction that could be inactivated by alkylation or proteolysis, was ATP-dependent, and was insensitive to the presence of a variety of uncouplers and ionophores.     

Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation.

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.     

Secretion in yeast: in vitro analysis of the sec53 mutant.

Of central importance to studying protein translocation via a combined genetic and biochemical approach is the in vitro analysis of yeast conditionally-lethal secretory mutants. Analysis of sec53 presented an opportunity not only to see if mutants could be examined in recently developed yeast in vitro translocation systems, but also to characterize further the nature of this mutant originally postulated to be defective in protein translocation. Membranes from sec53 were capable of translocating and glycosylating nascent prepro-alpha-factor in vitro in both sec53 and wild-type lysates at temperatures that were non-permissive for growth of the mutant cells. These results suggested that the Sec53 protein does not function directly in the translocation and glycosylation of prepro-alpha-factor. To examine this point further, we isolated membranes from sec53 cells that had been grown at the non-permissive temperature prior to disruption. In such cases, regardless of assay temperature, membranes from sec53 cells efficiently translocated but failed to glycosylate prepro-alpha-factor in vitro. The in vitro phenotype of sec53 could be mimicked by isolating rough microsomes from wild-type cells that had been grown for 1 h in the presence of tunicamycin. Together, these results demonstrate that sec53 is not defective in translocation, rather in assembly of the dolichol-oligosaccharide substrate needed for N-linked glycosylation.     

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.     

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.     

Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation

The large (L) envelope protein of the hepatitis B virus (HBV) has the peculiar capacity to form two transmembrane topologies via an as yet uncharacterized process of partial post-translational translocation of its pre-S domain across membranes. In view of a current model that predicts an HBV-specific channel generated during virion envelope assembly to enable pre-S translocation, we have examined parameters influencing L topogenesis by using protease protection analysis of wild-type and mutant L proteins synthesized in transfected cells. We demonstrate that contrary to expectation, all determinants, thought to be responsible for channel formation, are dispensable for pre-S reorientation. In particular, we observed that this process does not require (i) the helper function of the HBV S (small) and M (middle) envelope proteins, (ii) covalent dimer formation of envelope chains, or (iii) either of the three amphipathic transmembrane segments of L. Rather, the most hydrophobic transmembrane segment 2 of L was identified as a vital topogenic determinant, essential and sufficient for post-translational pre-S translocation. Cell fractionation studies revealed that pre-S refolding and thus the dual topology of L is established at the endoplasmic reticulum (ER) membrane rather than at a post-ER compartment as originally supposed. Together our data provide evidence to suggest that the topological reorientation of L is facilitated by a host cell transmembrane transport machinery such as the ER translocon.     

Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway

Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway. In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro. At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates. In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed. The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus. Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).     

Secretion in yeast: preprotein binding to a membrane receptor and ATP- dependent translocation are sequential and separable events in vitro

We have used a cytosol-free assay in which efficient translocation and signal peptide cleavage is achieved when the affinity-purified precursor of OmpA (proOmpA) is diluted out of 8 M urea into a suspension of yeast rough microsomes. This aspect of protein targeting and transport occurs in two discernible steps: (a) in the absence of ATP and cytosolic factors, the precursor binds to the membranes but is not translocated; (b) addition of ATP results in the translocation of the bound precursor and its processing to the mature form. The binding to microsomes of radiolabeled proOmpA is saturable and inhibited by the addition of unlabeled proOmpA but not by mature OmpA or other proteins. The binding of radiolabeled prepro-alpha-factor is also effectively competed by other preproteins, but not by mature ones. Scatchard analysis showed the Kd of proOmpA to be 7.5 X 10(-9) M. Binding is most likely protein mediated as treatment of the microsomes with the protease papain was found to be inhibitory. These results represent the first functional characterization of secretory protein precursor binding to membranes. Alkylation of the microsomes with NEM, washing the membranes with urea or using membranes from the (translocation) mutant ptll at the nonpermissive temperature, did not affect binding, but did eliminate the subsequent ATP-dependent translocation. The ability to subdivide translocation into individual reactions provides a more precise means of determining the membrane components involved in this process.     

Ratcheting in post-translational protein translocation: a mathematical model

We have developed a non-steady-state mathematical model describing post-translational protein translocation across the endoplasmic reticulum membrane. Movement of the polypeptide chain through the channel in the endoplasmic reticulum membrane is considered to be a stochastic process which is biased at the lumenal side of the channel by the binding of BiP (Kar2p), a member of the Hsp70 family of ATPases (ratcheting model). Assuming that movement of the chain through the channel is caused by passive diffusion (Brownian ratchet), the model describes all available experimental data. The optimum set of model parameters indicates that the ratcheting mechanism functions at near-maximum rate, being relatively insensitive to variations of the association or dissociation rate constants of BiP or its concentration. The estimated rate constant for diffusion of a polypeptide inside the channel indicates that the chain makes contact with the walls of the channel. Since fitting of the model to the data required that the backward rate constant be larger than the forward constant during early diffusion steps, translocation must occur against a force. The latter may arise, for example, from the unfolding of the polypeptide chain in the cytosol. Our results indicate that the ratchet can transport polypeptides against a free energy of about 25 kJ/mol without significant retardation of translocation. The modeling also suggests that the BiP ratchet is optimized, allowing fast translocation to be coupled with minimum consumption of ATP and rapid dissociation of BiP in the lumen of the ER. Finally, we have estimated the maximum hydrophobicity of a polypeptide segment up to which lateral partitioning from the channel into the lipid phase does not result in significant retardation of translocation.     

Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein

An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.     

Protein transport towards the thylakoid lumen: post-translational translocation in tandem

Many proteins found in the chloroplast are synthesized in the cytoplasm as precursor molecules containing transit peptides. Proteins targeted to the stroma must pass through the two envelope membranes to reach their destination. Proteins located in the chloroplast lumen also have to be transferred across the thylakoid membrane. That is, lumen proteins must cross three biological membranes in order to reach their final location. Recent evidence shows that the routing of plastocyanin towards the lumen involves two post-translational transport processes mediated by two different regions of the transit peptide and two different processing proteases. It is postulated that the genetic information for the plastocyanin precursor, which already contained a signal peptide, was transferred from the endosymbiont to the nucleus. Then a chloroplast-specific targeting-peptide was added.     

Secretion of invertase in mitotic yeast cells.

In mammalian cells intracellular transport is inhibited during mitosis. Here we show that in the yeast Saccharomyces cerevisiae secretion continues uninterrupted during mitosis. S. cerevisiae cells were arrested in mitosis by treating wild-type cells with the microtubule-inhibitor nocodazole, or by incubating a temperature-sensitive cell division cycle mutant (cdc16) at the restrictive temperature. Secretion of invertase into the periplasmic space was equally efficient in mitotic and in unsynchronized cells. Electron microscopy of nocodazole-treated mitotic wild-type cells revealed stretches of rough endoplasmic reticulum, strongly fenestrated Golgi cisternae and clusters of vesicles with the diameter of 30-90 nm. Secretion of invertase was inhibited in mitotic sec7 cells at the restrictive temperature, but continued at the permissive temperature. Sec7 is a mutant strain where intracellular traffic is blocked in unsynchronized cells in the Golgi complex at the restrictive temperature. Thus, the elements of the mitotic Golgi complex appear to be able to support intracellular traffic.     

Misplacement of the amino-terminal positive charge in the prepro-alpha-factor signal peptide disrupts membrane translocation in vivo

We have identified a series of mutations in the signal peptide of yeast prepro-alpha-factor which specifically attenuate translocation across the endoplasmic reticulum membrane in vivo. In prepro-alpha-factor-somatostatin hybrids, transposition of the amino-terminal tripeptide from wild-type NH2-Met-Arg-Phe to NH2-Met-Phe-Lys or NH2-Met-Phe-Arg causes a 45-75% reduction in the efficiency of membrane translocation. This is evidenced by the intracellular accumulation of unglycosylated, signal-containing precursors which are membrane-associated and are exposed to the cytosol. Surprisingly, abolition of the single positive charge by replacing arginine with phenylalanine has little effect on translocation into the endoplasmic reticulum. We conclude that the presence of an amino-terminal positive charge is not necessary for efficient targeting or translocation; however, misplacement by one position markedly disrupts translocation without affecting targeting. These mutations thus define an early stage of membrane interaction that is sensitive to local charge effects. Furthermore, our data suggest that post-translational translocation, signal cleavage, and core glycosylation of these polypeptides may occur to a significant extent in vivo.     

SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction.

Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs. Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells. We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide. However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex. Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.     

Secretion of human prolactin in vitro.

Cells from two human pituitary tumors were grown in capillary culture units and prolactin production was measured. A single unit could produce 3 mg of prolactin over a 4-month period. The cultured cells responded to TRH exposure by increasing their rate of prolactin secretion. Cultivation of cells in capillary units could be the method of choice for reducing the shortage of human hormones.     

Factors influencing expression and post-translational modification of recombinant protein C.

Factors affecting the production of recombinant human protein C were investigated. When recombinant cells producing human protein C were cultured with microcarrier in two different scales, we found that (1) as cells grew to confluence, specific productivity of the protein was decreased and that (2) the efficiency of gamma-carboxylation of the generated protein C was lower in a larger culture than in a smaller culture. Higher cell density was shown to influence the specific productivity unfavorably. On the other hand, the amount of oxygen supply as demonstrated by oxygen consumption rate in the Opticell culture system correlated well with the efficiency of gamma-carboxylation, suggesting that oxygen metabolism is somehow implicated in the post-translational modification of recombinant protein C.