We have previously reported that the expression in yeast of an integral membrane protein (p180) of the endoplasmic reticulum (ER), isolated for its ability to mediate ribosome binding, is capable of inducing new membrane biogenesis and an increase in secretory capacity. To demonstrate that p180 is necessary and sufficient for terminal differentiation and acquisition of a secretory phenotype in mammalian cells, we studied the differentiation of a secretory cell line where p180 levels had been significantly reduced using RNAi technology and by transiently expressing p180 in nonsecretory cells. A human monocytic (THP-1) cell line, that can acquire macrophage-like properties, failed to proliferate rough ER when p180 levels were lowered. The Golgi compartment and the secretion of apolipoprotein E (Apo E) were dramatically affected in cells expressing reduced p180 levels. On the other hand, expression of p180 in a human embryonic kidney nonsecretory cell line (HEK293) showed a significant increase in proliferation of rough ER membranes and Golgi complexes. The results obtained from knockdown and overexpression experiments demonstrate that p180 is both necessary and sufficient to induce a secretory phenotype in mammalian cells. These findings support a central role for p180 in the terminal differentiation of secretory cells and tissues.

Protein translocation into the yeast endoplasmic reticulum requires the transport of AT P into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC! gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SA C1 gene (saclA) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro-a-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a saclA strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Saclp was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the saclA strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the E R and into the Golgi complex in vivo. The collective data indicate an intimate role for Saclp in the transport of A TP into the E R lumen. S ECRETION in eucaryotic cells commences with the translocation of secretory proteins into the lumen of the ER. In the yeast Saccharomyces cerevisiae, different sets of genes have been defined, encoding cytosolic, luminal, and ER membrane proteins involved in this multistep process (for review see Nunnari and Walter, 1992; Schekman, 1994). Factors responsible for cotranslational targeting of nascent secretory proteins to the membrane, such as the signal recognition particle and its receptor, are not crucial for cell viability (Hann and Walter, 1991; Ogg et al., 1992). In contrast, the subset of genes whose products are believed to form the translocation site on the membrane are essential. Biochemical analysis in a reconstituted system indicated that translocation across the lipid bilayer is facilitated by two membrane protein complexes that interact in a dynamic manner. A putative translocation pore is formed by Sec61p, Ssslp, and Sbhlp (Esnault et aL, 1994; Panzner et al., 1995). The translocation process also requires the presence of an additional multisubunit complex consisting of Sec62p, Sec63p, Sec71p, Sec72p, and Kar2p, the yeast homologue of BiP (Brodsky and Address all correspondence to Dr. David Meyer, Department of Biological Chemistry, UCLA School of Medicine, Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90024-1737. Tel.: (310) 206-3122. Fax: (310) 206-5197. P. Mayinger's present address is Zentrum ftir Molekulare Biologie, Heidelberg, Germany. Schekman, 1993; Panzner et al., 1995). The importance of Kar2p in secretory protein translocation has also been shown genetically. In this case, a temperature-sensitive mutation in the KAR2 gene caused the accumulation of secretory protein precursors in the cytosol at the nonpermissive temperature (Vogel et al., 1990; Nguyen et al., 1991). Despite the rapid progress made in characterizing components required for translocation, our understanding of the driving force for the membrane passage of proteins is still limited. In a cotranslational mechanism, preproteins might be "pushed" across the membrane by elongation of the nascent chain itself. However, in yeast, where translocation can be uncoupled from translation (Toyn et al., 1988) and precursor proteins can be translocated posttranslationally into ER-derived vesicles in vitro (Hansen et al., 1986; Rothblatt and Meyer, 1986; Waters and Blobel, 1986), another driving force must be operating. Using a cytosol-free assay, it was shown that the posttranslational membrane transfer of purified precursors requires only the precursor, microsomal membranes, and ATP (Sanz and Meyer, 1988, 1989). Based on the results of in vivo (Vogel et al., 1990; Nguyen et al., 1991) and in vitro studies (Sanders et al., 1992; Brodsky and Schekman, 1993), Kar2p is the only ER protein identified to date that is directly involved in translocation in yeast and that uses A TE As a mechanism for translocation has been put forward in which preproteins are "pulled" across the mem© The Rockefeller University Press, 0021-9525/95/12/1377/10 $2.00 The Journal of Cell Biology, Volume 131, Numbe: 6, Part 1, December 1995 1377-1386 1377 on A uust 3, 2017 jcb.rress.org D ow nladed fom brane by ATP-dependent interactions with luminal chaperones (Simon et al., 1992; Glick, 1995), one can speculate that this represents the manifestation of the observed ATP requirement in the translocation process. Interactions of translocated chains with Kar2p are also essential for the proper folding of secretory proteins in the ER lumen and their further passage to the Golgi complex (Gething and Sambrook, 1992; Georgopoulos and Welch, 1993; Simons et al., 1995). A basic feature of such a mechanism is the continuous supply of ATP to the ER lumen. Recently a specific transport system was identified that allows efficient uptake of ATP into yeast ER (Mayinger and Meyer, 1993). When ATP uptake into microsomes was decreased below a critical level by specific inhibitors, coas well as posttranslational translocation of preproteins was blocked. In these studies, a reconstituted system was developed for the biochemical characterization of the ATP transporter. Using this assay, a component was purified that was shown to be the SAC1 gene product. Loss of SAC1 through gene deletion severely compromised translocation in vitro and subsequent intracellular transport steps in vivo. Incorporation of immunopurified Saclp into proteoliposomes enabled the reconstitution of high levels of ATP transport. These results make a strong case for a direct involvement of Saclp in the transport of ATP into the ER lumen, perhaps as the ATP transporter itself. Materials and Methods Reconstitution of A TP Transport into Proteoliposomes Yeast microsomes were prepared according to Rothblatt and Meyer (1986). The membranes were resuspended in a buffer containing 30 mg/ml Triton X-100, 150 mM Na2SO4 and 10 mM Tris-HC1, pH 7.4, at a concentration of 10 mg protein/ml. After 10 min incubation at 4°C the mixture was centrifuged at 100,000 g. The detergent concentration of the supernatant was adjusted to 60 mg/ml by the addition of Triton X-100. A sonicated mixture of phosphatidylcholine and cholesterol (10:1) was added to this solution resulting in a final lipid concentration of 24 mg/ml. After the solution became translucent it was supplemented with nucleotide counter substrates at a final concentration of 10 mM and the mixture was incubated for 30 min at 0°C. Liposomes were generated by removal of detergent accomplished by repeated passage over Bio Beads SM2 columns (Bio-Rad Laboratories, Hercules, CA) (1 g SM2 beads/30 mg Triton X-100). The proteoliposomes were separated from nonreconstituted material and solutes by gel filtration on a Sephadex G-100 column (Sigma Chemical Co., St. Louis, MO). A TP Transport Assay ATP uptake into yeast microsomes was measured as described previously (Mayinger and Meyer, 1993). Nucleotide exchange in proteoliposomes was determined as follows: 300 ~,l liposome suspension containing a suitable counter substrate (usually 10 mM ATP or ADP) was rapidly mixed with 25 ixM [14C]ATP at 25°C. The exchange was stopped at different time points by rapid filtration of 50-ix| aliquots using 100 mg wet Dowex (Sigma Chemical Co.) (8, chloride form, 100-200 mesh) in small glass columns. Free ATP is bound tightly by the strong anion exchange resin, while liposomes are not retained. The columns were washed once with 100 ixl 100 mM MOPS, pH 7.2. Radioactivity in the combined eluate and wash was quantified by scintillation counting. Nonspecific uptake was determined either by treatment of liposomes with 4,4'-diisothiocyano-2,2'-stibene disulfonic acid (DIDS), 1 an effective inhibitor of ATP transport, or by conducting the transport assay at 0°C. Both methods gave approximately the 1. Abbreviat ions used in this paper: CPY, carboxypeptidase Y; DIDS, 4,4'diisothiocyano-2,2'-stibene disulfonic acid; pp-aF, prepro-a-factor; ppCPY, prepro--carboxypeptidase Y. same level of background. All transport assays were performed in the presence of carboxyatractyloside, a specific inhibitor of mitochondrial ATP transport, to rule out any influence of mitochondrial contamination. Purification of ATP Transport Activity Membranes (10 mg/ml final concentration) were extracted with 3 % Triton X-100 and 150 mM Na2SO 4 and 10 mM Tris-HC1, pH 7.4. The extract was mixed with a pasty suspension of hydroxyapatite (100 rag/rag protein). After centrifugation the supernatant was applied to an ATP agarose column (2.5 ml/ml supernatant). The column was washed with 10 ml buffer A (500 mM NaC1, 1% Triton X-100, 10 mM Tris-HCl, pH 7.4) and with 10 ml buffer B (50 mM NaCl 1% Triton X-100, 10 mM Tris-HC1, pH 7.4). Then the column was eluted with 10 ml buffer B supplemented with 3 mM A T E The eluate was concentrated on a centriconl0 spin column (Amicon, Beverly, MA). The concentrated fractions were analyzed by SDS-PAGE or reconstituted into proteoliposomes and assayed for ATP transport as described above. Microsequence Analysis The ATP agarose eluate was analyzed by SDS-PAGE using 10% polyacrylamide gels. After staining and destaining, bands of interest were cut out and the gel pieces were washed twice for 20 min with 150 ill 50% acetonitrile to dehydrate the gel. The shrunken gel pieces were placed on parafilm and dried for an additional 15-25 min, The dried pieces were incubated overnight in 100 ~,l 80% formic acid containing 2 mg cyanogen bromide. To remove formic acid and excess cyanogen bromide the gel pieces were treated by three 20-min washes in I ml water followed by lyophilization. Then the pieces were incubated

Expression of the 180-kDa canine ribosome receptor in Saccharomyces cerevisiae leads to the accumulation of ER-like membranes. Gene expression patterns in strains expressing various forms of p180, each of which gives rise to unique membrane morphologies, were surveyed by microarray analysis. Several genes whose products regulate phospholipid biosynthesis were determined by Northern blotting to be differentially expressed in all strains that undergo membrane proliferation. Of these, the INO2 gene product was found to be essential for formation of p180-inducible membranes. Expression of p180 in ino2Delta cells failed to give rise to the p180-induced membrane proliferation seen in wild-type cells, whereas p180 expression in ino4Delta cells gave rise to membranes indistinguishable from wild type. Thus, Ino2p is required for the formation of p180-induced membranes and, in this case, appears to be functional in the absence of its putative binding partner, Ino4p.

Targeting of the minus-end directed microtubule motor cytoplasmic dynein to a wide array of intracellular substrates appears to be mediated by an accessory factor known as dynactin [1-4]. Dynactin is a multi-subunit complex that contains a short actin-related protein 1 (Arp 1) filament with capZ at the barbed end and p62 at the pointed end [5]. The location of the p62 subunit and the proposed role for dynactin as a multifunctional targeting complex raise the possibility of a dual role for p62 in dynein targeting and in Arp1 pointed-end capping. In order to gain further insight into the role of p62 in dynactin function, we have cloned cDNAs that encode two full-length isoforms of the protein from rat brain. We found that p62 is homologous to the nuclear migration protein Ropy-2 from Neurospora [6]; both proteins contain a zinc-binding motif that resembles the LIM domain of several other cytoskeletal proteins [7]. Overexpression of p62 in cultured mammalian cells revealed colocalization with cortical actin, stress fibers, and focal adhesion sites, sites of potential interaction between microtubules and the cell cortex [8,9]. The p62 protein also colocalized with polymers of overexpressed wild-type or barbed-end-mutant Arp1, but not with a pointed-end mutant. Deletion of the LIM domain abolished targeting of p62 to focal-adhesion sites but did not interfere with binding of p62 to actin or Arp1. These data implicate p62 in Arp1 pointed-end binding and suggest additional roles in linking dynein and dynactin to the cortical cytoskeleton.

Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin's most abundant component, Arp1alpha was transiently overexpressed in mammalian cells. Arp1alpha overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1alpha in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1alpha that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1alpha proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1alpha, NuMA localization remained unaffected. Although expression of mutant Arp1alpha proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1alpha interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1alpha mutants. We conclude that the amino acids altered in the Arp1alpha mutant proteins participate in stabilizing interactions between overexpressed Arp1alpha and components of the endogenous dynactin complex as well as the NuMA protein.

Review Articles| May 07 1999 Assignment of β-centractin (CTRN2) to human chromosome 2 bands q11.1→q11.2 with somatic cell hybrids and in situ hybridization Subject Area: Genetics S.H. Elsea; S.H. Elsea Departments of Neurology, and Search for other works by this author on: This Site PubMed Google Scholar I.B. Clark; I.B. Clark Department of Biological Chemistry, UCLA School of Medicine and the Molecular Biology Institute, Los Angeles CA (USA) Search for other works by this author on: This Site PubMed Google Scholar R.C. Juyal; R.C. Juyal Departments of Neurology, and Search for other works by this author on: This Site PubMed Google Scholar D.J. Meyer; D.J. Meyer Department of Biological Chemistry, UCLA School of Medicine and the Molecular Biology Institute, Los Angeles CA (USA) Search for other works by this author on: This Site PubMed Google Scholar D.I. Meyer; D.I. Meyer Department of Biological Chemistry, UCLA School of Medicine and the Molecular Biology Institute, Los Angeles CA (USA) Search for other works by this author on: This Site PubMed Google Scholar P.I. Patel P.I. Patel Departments of Neurology, and Molecular and Human Genetics, Baylor College of Medicine, Houston TX; Search for other works by this author on: This Site PubMed Google Scholar Cytogenetics and Cell Genetics (1999) 84 (1-2): 48–49. https://doi.org/10.1159/000015211 Article history Published Online: May 07 1999 Content Tools Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation S.H. Elsea, I.B. Clark, R.C. Juyal, D.J. Meyer, D.I. Meyer, P.I. Patel; Assignment of β-centractin (CTRN2) to human chromosome 2 bands q11.1→q11.2 with somatic cell hybrids and in situ hybridization. Cytogenetics and Cell Genetics 1 July 1999; 84 (1-2): 48–49. https://doi.org/10.1159/000015211 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsCytogenetic and Genome Research Search Advanced Search This content is only available via PDF. 1999Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Article PDF first page preview Close Modal You do not currently have access to this content.

Ribosome binding to the endoplasmic reticulum has been traditionally studied using an in vitro assay in which potential ribosome receptors have been purified, incorporated into synthetic liposomes, and tested for activity. One such receptor (180 kDa; “p180”) has been shown to bind ribosomes with high affinity in such a system when purified to homogeneity. This result has been challenged by data generated in other laboratories, and as a result, doubt has lingered as to the authenticity of p180 as a ribosome receptor. The contribution of the major difference between these studies, the lipid composition of the liposomes used in the in vitro assays, was assessed when identical fractions of rough endoplasmic reticulum-specific membrane proteins were incorporated into liposomes composed of only phosphatidylcholine (as used in other laboratories), a 50:50 mix of phosphatidylcholine and phosphatidylserine (as used in our original studies), or lipids derived from canine pancreatic microsomes (as a physiologically relevant control). The presence of PS was found to be crucial for the incorporation into and ribosome binding activity of p180 in liposomes. These observations are compatible with published studies on the importance of acidic phospholipids in ribosome binding to intact microsomes and reconcile the apparently conflicting in vitro results surrounding the assignment of p180 as a ribosome receptor.

Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha- factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed.

A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned and sequenced. The deduced primary structure indicates three distinct domains: an NH2-terminal stretch of 28 uncharged amino acids representing the membrane anchor, a basic region (pI = 10.74) comprising the remainder of the NH2-terminal half and an acidic COOH- terminal half (pI = 4.99). The most striking feature of the amino acid sequence is a 10-amino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem without interruption in the NH2-terminal positively charged region. We postulate that this repeated sequence represents a ribosome binding domain which mediates the interaction between the ribosome and the ER membrane. To substantiate this hypothesis, recombinant full-length ribosome receptor and two truncated versions of this protein, one lacking the potential ribosome binding domain, and one lacking the COOH terminus, were expressed in Saccharomyces cerevisiae. Morphological and biochemical analyses showed all proteins were targeted to, and oriented correctly in the ER membrane. In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or one lacking the COOH-terminal domain were able to bind two to four times as many human ribosomes as control membranes lacking a recombinant protein or microsomes containing a receptor lacking the NH2-terminal basic domain. Electron micrographs of these cells revealed that the expression of all receptor constructs led to a proliferation of perinuclear ER membranes known as "karmellae." Strikingly, in those strains which expressed cDNAs encoding a receptor containing the putative ribosome binding domain, the induced ER membranes (examined in situ) were richly studded with ribosomes. In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding domain were uniformly smooth and free of ribosomes. Cell fractionation and biochemical analyses corroborated the morphological characterization. Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding.

As part of our ongoing efforts to understand the functional role of vertebrate centractins, we have identified a new member of the actin-related family of proteins in the yeast Saccharomyces cerevisiae using a PCR-based approach. Consistent with the current nomenclature for actin-related proteins in yeast, we propose to denote this locus ACT3. The primary amino acid sequence of Act3p is most similar to canine and human alpha-centractin (73% similarity/54% identity). The sequence of a genomic clone indicates ACT3 lies adjacent to and is transcribed convergently with respect to FUR1 on chromosome VIII. Molecular genetic analysis indicates ACT3 is represented by a single gene from which the corresponding mRNA is expressed at a low level compared to ACT1. Tetrad analysis of heterozygotes harboring a TRP1 replacement of the ACT3-coding region indicates ACT3 is nonessential for growth under normal conditions and at extremes of temperature and osmolarity. However, growth at 14 degrees C indicates a spindle orientation defect similar to phenotypes recently described for yeast harboring mutations in actin, tubulin, or cytoplasmic dynein. Taken together, our data suggest that ACT3 is the S. cerevisiae homologue of vertebrate centractins.

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 × 10(−5) M. ATP transport into ER vesicles was specifically inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3′‐O‐(4‐benzoyl)benzoyl‐ATP (Bz2‐ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2‐ATP blocked protein translocation in vitro measured co‐ as well as post‐translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS‐treated membranes. These results demonstrate that translocation requires a DIDS and Bz2‐ATP‐sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.

The molecular mechanism by which precursors of secretory proteins traverse the membrane of the endoplasmic reticulum (ER) is still poorly understood. Of particular interest is the question how this process is linked energetically to the hydrolysis of ATP. When post-translational modes of translocation in yeast and in mammalian ER were examined, one role for ATP could be attributed to the function of cytosolic stress factors, which maintain nascent polypeptides in a translocation competent state (Meyer, 1991). Recently, an additional ATP requirement of the translocation step at the membrane level has been found for both systems. In yeast it was shown that ATP is required for the membrane transfer of preproteins, bound to receptor sites on the ER membrane (Sanz and Meyer, 1989). With regard to this finding basically two models can be proposed how ATP is used for ER-rnembrane transfer of preproteins.