Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Oehlp. Several constructs included a Kex2p cleavage site between the Ochlp and invertase moieties to monitor transit to the Kex2p-containing TGN. Cells expressing an Ochlp-invertase fusion do not secrete invertase, but those expressing an Ochlp-Kex2p site-invertase fusion protein secrete high levels of invertase in a Kex2pdependent manner. The Ochlp-Kex2p site-invertase fusion protein is cleaved with a half-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medialand trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Ochlp-HA moiety, irrespective of whether it is generated by cleavage of the fusion protein in the TGN or synthesized de novo, is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN. At steady state, de novo--synthesized and TGN-generated HA epitope-tagged Ochlp reside in a compartment with a buoyant density identical to that of wild-type Ochlp and distinct from that of the vacuole or the TGN. Finally, ochl null cells that express an Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Ochlp activity. These results lead us to propose a model for Ochlp-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.
T HE eukaryotic secretory pathway consists of a set of membrane-bound compartments that are involved in delivery of newly made proteins to their final destinations. The hub of this pathway is the Golgi apparatus, which is involved in the posttranslational modification and sorting of the proteins that pass through it. The Golgi is composed of several topologically distinct cisternae, each of which contains a unique set of resident proteins. These proteins must resist dispersion by the large flux of vesicular traffic to and from the Golgi apparatus. This process is as important as the initial delivery of proteins to this organelle, yet it is not well understood. For example, it is not known whether newly made Golgi proteins simply cease their forward movement upon reaching the correct cisternae, or whether they are localized by a more dynamic mechanism involving recycling through later compartments.
Compared to the mechanism of ER protein retention, much less is known about the mechanism of protein retention in the Golgi apparatus. Although the scattered Golgi apparatus of Saccharomyces cerevisiae is morphologically distinct from its stacked mammalian counterpart, it is quite similar biochemically, and therefore provides an excellent model system for analysis of Golgi protein retention. Several lines of evidence indicate that the yeast Golgi, like that of mammalian cells, is composed of distinct compartments. For example, the sequential maturation of oligosaccharides in the yeast Golgi requires the SEC18 gene product (yeast N-ethylmaleimide-sensitive fusion protein, NSF) at multiple stages, indicating that the modification enzymes are sequestered in functionally and topologically distinct compartments (31) . Localization of Golgi resident proteins by indirect immunofluorescence (26) and subcellular fractionation (9, 11, 18, 28, 105) also reveals that different Golgi enzymes reside in separable compartments. Similarly, transport of pro-a-factor from the ER to the Golgi in vitro results in only the earliest glycosyl modification, indicating that the glycosyl transferases used at later steps are not contained within the acceptor compartment (4, 34) . In sum, these and other studies indicate that the yeast Golgi can be subdivided into at least four compartments. The ER-proximal, or cis, compartment contains the initiating ~t-l,6-mannosyltransferase (ManT), Ochlp (28, 65) , which is responsible for addition of the first mannose residue to the core oligosaccharides obtained in the ER (64, 80) . The next, or medial, compartment contains the elongating o~-l,6-ManT activity (28) . The terminal et-l,3-ManT, encoded by MNN1 (77) , is contained within the equivalent of the trans-Golgi and TGN (31, 33) . The TGN, which is the most distal Golgi compartment, also contains the Kex2p, Kexlp, and DPAP A proteases (11, 18, 25, 31) .
Most known residents of the cis-, medial-, and trans-Golgi are type II IMPs, with their amino termini in the cytoplasm and their carboxy termini in the Golgi lumen. (A recently described exception is Emp47, which is a Type I IMP of the yeast medial-Golgi [88] ). In contrast, TGN residents are generally type I IMPs with the opposite topology. Much effort has been directed towards identifying the "Golgi retention signal" of early type II proteins. Many studies indicate that the transmembrane domain is critical (2, 14, 32, 39, 57, 89, 98, 99, 111) with some researchers finding that the adjacent regions are also involved (15, 19, 58, 59, 62, 66, 83, 100, 104) . However, several groups have argued that retention is conferred by multiple domains of the molecule (3, 7, 12, 32, 54, 90) . For example, in yeast, the transmembrane domain of Mnnlp can localize a heterologous protein to the Golgi; nevertheless, a soluble form of Mnnlp, completely lacking a transmembrane domain, is also localized to the Golgi (32) . Thus, there may be multiple signals involving cytoplasmic, membrane-spanning, and luminal domains that act together to affect the proper localization of Golgi proteins.
In contrast to the complex retention signals of type II cis, medial, and trans residents, the retention signals of three type I residents of the yeast TGN are well defined: they are located in the carboxy-terminal cytoplasmic tails, contain critical aromatic residues, and are similar to the signals within mammalian cell surface receptors that medi-ate clustering into clathrin-coated pits (107) . Elimination of the cytoplasmic tails of the TGN proteases DPAP A, Kex2p, or Kexlp destroys retention and results in transport to the vacuole (16, 69, 106) . In addition, loss of clathrin function in yeast results in mislocalization of Kex2p and DPAP A to the plasma membrane (91) . It was suggested, therefore, that clathrin binding to the cytoplasmic tails of Kex2p or DPAP A was necessary for either direct retention or recycling of late Golgi enzymes (91) .
Localization of the type I proteins to the TGN and type II proteins to the earlier compartments appears to occur via different mechanisms. First, clathrin may not be directly required for localization of early Golgi proteins because GDPase, a type II early Golgi protein, is not mislocalized in a ctathrin mutant (92) . In contrast, Mnnlp, a type II enzyme that seems to have dual citizenship in both the trans-Golgi and TGN of yeast, is mislocalized in a clathrin mutant (33) . However, this effect is not mediated through the cytoplasmic tail of Mnnlp (33) , and it may be due to a more indirect effect on TGN structure or function in clathrin mutants. Second, the early and late Golgi retention mechanisms differ in their saturability. For example, overexpression of the type I proteins Kex2p or DPAP A causes a significant fraction of the protein to mislocalize to the vacuole (69, 106) , whereas the Golgi seems to be able to readily localize overexpressed Mnnlp, which is a type II protein (32) . Behavior of Mnnlp is more similar to mammalian glycosyltransferases, which upon overexpression are predominately correctly localized, although ER accumulation is sometimes observed (62, 66, 97) . Finally, early type II and late type I retention systems appear to be distinct because overexpression of Mnnlp does not alter the turnover of Kex2p, suggesting that the proteins are not competing for a common component (33) .
Several mechanistic models have been proposed for Golgi protein localization (10, 55, 67) . Probably the most widely held view, first proposed by Swift and Machamer (97) , is that Golgi proteins form aggregates that are too large to enter forward-moving transport vesicles and are thus retained (53, 68, 90, 97, 104) . This model requires that aggregation be induced at a particular point during Golgi transit, perhaps by some physiochemical property of particular Golgi subcompartments. A related model, called the "kin recognition model" (67, 68) , posits that different resident proteins of a particular cisternae interact to form large heterooligomers that restrict forward movement, possibly by anchoring to a Golgi matrix (96) . Yet a third model is based on two observations: first, that Golgi protein transmembrane domains are shorter, in general, than those of plasma membrane proteins (10) , and second, that a cholesterol concentration gradient seems to exist in the mammalian secretory pathway (72) , with the ER being relatively depleted and the plasma membrane being relatively enriched. Cholesterol tends to thicken lipid bilayers, hence the proposal that proteins transit the secretory pathway until their transmembrane domains "fit" the thickness (35) of the surrounding bilayer. Premature retention or continued movement forward would be energetically unfavorable due to the hydrophobic effect. One of the features common to all these models is that once a protein reaches its correct subcompartment within the Golgi, it is postulated to cease its forward movement and be retained.
We have investigated the localization mechanism of Ochlp, a cis-Golgi ManT, using a series of influenza virus hemagglutinin (HA) epitope-tagged Ochlp-invertase fusion proteins (see Fig. 1 ). In contrast to most current models, we have found that the Ochlp fusion proteins do not efficiently halt their forward movement upon encountering the cis compartment. Instead, they move to later Golgi compartments, at least as far as the TGN, after which they return to the cis compartment. Our results indicate that yeast possesses a retrograde transport pathway from the TGN to the cis compartment, and furthermore, that localization of HA epitope-tagged Ochlp to the cis-Golgi at steady state is due, at least in part, to recycling from later Golgi cisternae.
The Escherichia coli strains used were XL1-Blue (supE44 thi-1 lac endA1 gyrA96 hsdR17 relA1 [ (this study). Yeast strains were grown on yeast extract, peptone, and dextrose or on synthetic dextrose (SD) medium supplemented as necessary (81) .
To create plasmid pSEC18 (URA3, CEN), the BamHI-HindIII SEC18containing fragment from plasmid pSEY8-SEC18 (2 ~m) (22) , kindly provided by S. Emr (University of California, San Diego), was subcloned into the yeast CEN-containing shuttle vector pRS416 (95) that had also been digested with BamHI and HindIII. Plasmid pKEX2-HA (TRP1, CEN) was made by snbcloning the KEX2-HA-bearing SalI/Eagl fragment from pSN218 (70), kindly provided by T. Stevens (University of Oregon, Eugene), into the yeast CEN-containing shuttle vector pRS414 (95) that had been digested with SalI and EagI. The KEX2 gene on pKEX2-HA contains the sequence encoding three repeated copies of the 9-amino acid epitope (YPYDVPDYA) from HA protein (110) . Plasmid pSH113 was created by digesting pBL-OCH1, generously provided by Y. Jigami (National Institute of Bioscience and Human Technology, Tsukuba, Japan) (65), with XcmI followed by incubation with T4 DNA polymerase to yield blunt ends. The linearized plasmid was then digested with NsiI and ligated to a SmaI-PstI LEU2 fragment from pMR2253, kindly provided by M. Plasmid pO, which encodes Ochlp, was created by inserting the 2.6-kb XhoI-HindIII OCH1 fragment from pBL-OCH1 into pRS416. Plasmid pO-HindIII was made by introducing a HindIII site at the codon for amino acid 472 (of the 480 amino acid Ochlp) by unique site elimination site-directed mutagenesis (20) . Plasmid pOI, which encodes the first 472 amino acids of Ochlp fused in frame to amino acid 2 of mature invertase, was created by ligating the SUC2-containing HindlII-PvuII fragment from plasmid pSEY306 (44) to pO-HindIII that had been digested with HindIII and SaclI. The amino acid sequence in single letter code at the Ochlp-invertase fusion ioint encoded by pOI is KEAST (where K is amino acid 470 of Ochlp and T is amino acid 2 of mature invertase). Plasmid pOKI encodes, at the junction between Ochlp and invertase, six amino acids from pro-a-factor (MYKREA) that contain the dibasic recognition site for the Kex2 protease. Plasmid pOKI was created by site-directed mutagenesis of the pOI plasmid using a mutagenic oligonucleotide that spanned OCH1 and SUC2. In one round of mutagenesis, the HindIII site at the junction of OCH1 and SUC2 was removed, amino acids 472-478 of Ochlp were restored, the Kex2 (MYKREA) cleavage site was added, and a NheI site was created. The sequence present at the fusion junction encoded by pOKI is: G (amino acid 478 of Ochlp) AS (encoded by the NheI site) MYKREA (Kex2p cleavage site) M (amino acid 1 of mature invertase).
To construct vectors encoding HA epitope-tagged proteins, we inserted a sequence encoding three repeated copies of the epitope (YPYDVP-DYAG) from HA (110) . First, a NheI site was added to the carboxy terminus of Ochlp by site-directed mutagenesis of plasmid pO resulting in the replacement of amino acids 479 and 480 (HK) with the amino acids AS in pO-NheI. To make pOH, a 100-bp fragment encoding the triple HA epitope sequence was removed from plasmid pMR2654 (kindly provided by M. Rose) by digestion with XbaL The XbaI fragment was ligated into the NheI site of pO-NheI. The sequence at the carboxy terminus of Ochlp encoded by pOH is G (amino acid 478 of Ochlp) AR YPYDVPDYAG YPYDVPDYAGSYPYDVPDYASS. Similarly, the same 100-bp HA epitope-encoding XbaI fragment was ligated into the NheI site of plasmid pOKI to create pOHKI (URA3). The fusion junction between Ochlp and invertase encoded by pOHKI is G (amino acid 478 of Ochlp) AR YPYD-VPDYAG YPYDVPDYAG SYPYDVPDYASS MYKREA (cleavage site) M (amino acid 1 of mature invertase), pOHKI (TRP1) was created by digesting pOHKI (URA3) with XhoI and SaclI and ligating the OCH1-SUC2--containing fragment into pRS414 (TRP1 CEN) (95) that had been digested with XhoI and Sacll. Plasmid pOKH, created by site-directed mutagenesis of plasmid pOH, encodes an Ochlp fusion protein whose carboxy terminus is appended with the Kex2 cleavage site followed by the triple HA epitope. The sequence at the carboxy terminus of Ochlp encoded by pOKH is G (amino acid 478 of Ochlp) SS MYKREA (cleavage site) YPYDVPDYAG YPYDVPDYAG SYPYDVPDYASS. All mutations were verified by DNA sequence analysis by the dideoxy method (85) .
E. coli transformations were performed using the method of Hanahan (36) . Yeast transformations were performed by the method of Schiestl and Gietz (87) with the modifications of Eible (23) . To create the Aochl strain, SHY101, a XhoI-HindIII ochl::LEU2-containing fragment from plasmid pSHll3 (described above) was integrated into diploid strain SHY100 (a/a, ura3-52/ura3-52, ade2-1OllADE2, leu2-3,112/leu2-3,112, TRP1/trplA1) by one-step gene replacement (82) . After sporulation, temperature-sensitive Leu ÷ haploid segregants were isolated that contained a complete deletion of the OCH10RF. Deletion of OCH1 was confirmed by PCR (84) using oligonucleotide primers flanking the deletion. SHY102 was created from SEY6210 by one-step gene replacement of KEX2 with the BamHI fragment containing the kex2A2::HlS3-s allele from plasmid pKX::HIS3-s, kindly provided by R. Fuller (University of Michigan, Ann Arbor). Deletion of KEX2 was confirmed by a decrease in mating efficiency of the null strain. SHY103 was created from SEY6210 by one-step gene replacement of PEP4 with the BamHI fragment containing the pep4A:.'LEU2 allele from plasmid pTS17 (kindly provided by T. Stevens). The deletion of PEP4 was confirmed by the inability of the cells to proteolytically process carboxypeptidase Y to its mature form (27, 47) .
Invertase activity was determined by the method of Goldstein and Lampen (29) with the modifications for measurement of periplasmic activity presented in Johnson et al. (44) .
The gene fragment ranging from the Hpal site to the NsiI site of OCH1 was cloned behind the gene encoding the maltose binding protein in the vector pMAL-c2 (New England Biolabs, Beverly, MA). The recombinant protein that included amino acids 71 to 480 of Ochlp was expressed in E.
coli and purified on an amylose column according to the manufacturer's direction. The purified recombinant protein was used for immunization of a rabbit according to standard procedures (37) .
Yeast protein extracts for immunoblotting were prepared from 1 ml of midlogarithmic phase (OD600 am = 0.5--1.0) cells grown in SD media containing appropriate supplements. Cells were washed in water and then resuspended in 0.2 ml 5% TCA. Glass beads (0.45-p.m diam) were added to about three-fourths of the total sample volume. Cells were lysed by vortexing for five 1-min periods, alternating with l-rain incubations on ice. The lysates were removed from the beads and centrifuged for 15 min in a microfuge at 4°C. TCA-precipitated protein was resuspended in Laemmli sample buffer (50) , heated at 100°C for 4 min, and separated on an 8% SDS-polyacrylamide gel. Immunoblots, which were developed with the enhanced chemiluminescence detection kit (Amersham Corp., Arlington Heights, IL), were performed according to standard methods (37) . HA epitope-tagged Ochlp and Kex2p were detected with 12CA5 hybridoma serum-free culture supernatant at a dilution ranging from 1:250 to 1:1000. Mouse monoclonal antibody 10D7-A7-B2 (21) , which recognizes the yeast vacuolar H+-ATPase 100 kD subunit, was used at a concentration of 250 ng/ml (Molecular Probes, Eugene, OR).
Yeast were grown in SD media lacking uracil, cysteine, and methionine at 30°C (unless otherwise indicated) to an OD600 nm = 0.5-1.0. 30D U of cells per time point were washed in SD lacking uracil, cysteine, and methionine and resuspended in 1 ml of the same media. Cells were pulse labeled with 150 p~Ci of Tran~SS-label (ICN Radiochemicals, Irvine, CA) per 3 OD U of ceils for 2 min at 30°C (unless otherwise noted). Labeling was quenched by addition of a 25 × chase solution (250 mM methionine, 250 mM cysteine) to a final concentration of IX. The chase was stopped by addition of NaN3 to 10 mM and cycloheximide to 1OO p~g/ml on ice. The cells were centrifuged, and the pellets were resuspended in 1OO p.l of bead buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCI, 2 mM EDTA, 1% SDS, and 1 mM PMSF). Glass beads (0.45-1xm diam) were added to about three-fourths of the total sample volume, and cells were disrupted by vortexing for five l-rain intervals separated by l-rain cooling periods on ice. The lysate was removed, and the beads were washed with 150 ttl of bead buffer. The lysate and wash were combined and incubated at 100°C for 4 min, centrifuged (12,OO0 g) for 10 rain to remove insoluble material, and added to 750 ~1 of immunoprecipitation (IP) dilution buffer (60 mM Tris-HCI, pH 7.4, 190 mM NaC1, 6 mM EDTA, 1.25% Triton X-1OO, and I mM PMSF). To precipitate HA epitope-tagged protein, 2OO I~1 per 3 OD U of cells of 12CA5 hybridoma serum-free culture supernatant was added. To precipitate CPY, 0.6 ixl of rabbit polyclonal anti-CPY per 3 OD U of cells were added. Antibody to CPY was a gift from S. Emr. Immunoprecipitation of invertase used 9 p.l of rabbit polyclonal anti-invertase, provided by M. Rose, per 3 OD U of cells. 20 p~l of a 50% slurry of protein A~Sepharose CL-4B (Pharmacia, Piscataway, NJ) in IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaC1, 5 mM EDTA, 1% Triton X-1OO, 0.2% SDS) was added, and the immune complexes were allowed to form overnight at 4°C while slowly mixing. Sepharose-bound immune complexes were pelleted (12,000 g, 1 rain) and washed sequentially in 1 ml each of high salt wash buffer (50 mM Tris-HCl, pH 7.4, 500 mM NaCI, 1% Triton X-1OO, 0.2% SDS), IP buffer, and detergent-free buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaC1, 5 mM EDTA). Immunoprecipitated protein was resuspended in Laemmli buffer (50) , incubated at 100°C for 4 min, and separated on an 8% SDS-polyacrylamide gel. Gels were fixed in 25% isopropanol, 10% acetic acid for 15 min, treated with Amplify (Amersham Corp.) for 15 min, dried, and exposed to film (X-AR5; Eastman-Kodak Co., Rochester, NY) at -70°C.
Antigens to be reimmunoprecipitated with a second antibody were dissociated from the first antibody by incubation at 1OO°C for 10 rain in the presence of 100 ~1 of 1% SDS. 9OO/~1 of IP dilution buffer was added to the eluted antigen. 5 p.l of polyclonal antisera specific to tx-l,6--or et-l,3linked mannose residues were added, and the immune complexes were allowed to form overnight at 4°C with gentle mixing. Complexes were washed and processed as described above. The linkage-specific antibodies (5) were provided by R. Schekman (University of California, Berkeley).
Yeast strain SHY102 (Akex2) expressing HA epitope-tagged KEX2 on a TRP CEN plasmid was transformed with either the pOH (URA3) or pOHKI (URA3) plasmid. The two strains were grown in 250 ml of SD lacking uracil and tryptophan at 30°C to an OD600 nm of 0.5-0.8, harvested by centrifugation, and washed two times in spheroplast buffer (50 mM Tris-HCl, pH 7.5, 1.2 M sorbitol, 10 mM NAN3, 40 mM 13-mereaptoethanol). Cells were resuspended in spheroplast buffer at 40 OD U/ml. Zymolyase 100T (ICN, Costa Mesa, CA) was added to 150 Ixg/ml, and cells were incubated at 30°C for 30 min with occasional mixing. The spheroplasts were washed two times in spheroplast buffer and resuspended in 0°C lysis buffer (20 mM Hepes/KOH, pH 7.4, 100 mM K-acetate, 5 mM Mg(acetate), 1 mM EDTA, 1 mM Dq'T) plus 2 mM EGTA, 1 mM PMSF, 5 mM 1,10-phenanthroline, 2 IxM pepstatin A, 2 p.g/ml aprotinin, 0.5 Ixg/ml leupeptin to the same volume in which they were spheroplasted. Cells were lysed in a 7-ml Dounce homogenizer (25 strokes of "B" pestle; Kontes Glass Co., Vineland, NJ) on ice, and the lysates were centrifuged in a rotor (SA600; Sorvail Instruments Div., DuPont Co., Newton, CT) at 4°C for 3 rain at 3,300 rpm (1OO0 g). The resulting supernatants (S1) were centrifuged in a Sorvail SA6OO rotor at 4°C for 15 rain at 10,OO0 g to generate pellet (P10) and supernatant (S10) fractions. The $10 fractions (1.3 ml) were loaded onto the tops of 10.5-ml continuous 25--40% sucrose (wt/wt) gradients and centrifuged in a rotor (SW4I; Beckman Instruments, Fullerton, CA) at 40,000 rpm at 4°C for 18 h. 15 0.75-ml fractions were collected starting from the bottom of the gradient.
To localize wild-type Ochlp, SEY6210 cells were grown in SD media lacking cysteine and methionine at 30°C to an OD~00 of 0.5-1.0. 20 OD U of cells were washed in SD lacking cysteine and methionine and resuspended in 12 ml of the same media. Cells were pulse labeled with 50 g, Ci of Tran35S-label per OD of cells for 2 min at 30°C. Labeling was quenched by addition of a 25x chase solution (250 mM methionine, 250 mM cysteine) to a final concentration of 1 ×. Cells were incubated at 30~C for 60 min after which the chase was stopped by addition of NaN 3 to 10 mM and cycloheximide to 100 p~g/ml on ice. An additional 60 OD U of unlabeled SEY6210 cells that had been washed two times in spheroplast buffer were added to the labeled cells. The cells were then spheroplasted and lysed, and the protein was fractionated on a 25-40% sucrose gradient as described above. The protein in 0.65 ml of each fraction was immunoprecipitated using 1 ixl of the rabbit polyclonal anti-Ochlp antisera per fraction. Immunoprecipitations were performed as described above.
Most models of protein retention within the Golgi apparatus propose a static mechanism whereby proteins fail to move forward upon reaching their resident compartment (10, 67), although retrieval mechanisms have been discussed (32, 40, 56) . In the light of recent data suggesting a recycling component to the localization mechanisms of ER (28, 74, 101) and TGN resident proteins (108), as well as a type I IMP in the medial-Golgi of yeast (88), we thought it was possible that localization of type II IMPs within the Golgi stacks might also involve retrieval of proteins that have escaped to more distal compartments. To examine this question, we chose to use Ochlp because its activity represents the first post-ER enzymatic event required for synthesis of outer-chain mannose linkages on yeast secretory proteins, and thus Ochlp must reside in a very early Golgi compartment (64, 80) . This cis-Golgi localization has recently been confirmed by Gaynor et al. (28) . To monitor the trafficking of Ochlp, it was fused to the reporter enzyme invertase, which is encoded by the SUC2 gene (Fig. 1) . Secretion of invertase into the yeast periplasm is necessary for cells to use sucrose as a carbon source (13) . A recognition site for the Kex2 protease was included at the junction of Ochlp and invertase to monitor passage of the fusion protein into the Kex2p compartment, or TGN. The amino acid sequence encoding the Kex2 cleavage site (MYKREA) was derived from the MFal gene (49) . MF~I encodes a precursor protein that transits the secretory pathway and is proteolytically processed by several enzymes in the TGN to yield mature a-factor pheromone, which is secreted from the cell (27, 78) . The Kex2p endopeptidase cleaves on the carboxyl side of the KR sequence found in the a-factor peptide (1, 45) .
We reasoned that if a static retention mechanism exists for Ochlp, then the Ochlp-Kex2 site-invertase fusion protein would remain in the cis-Golgi. This anchoring would result in a Suc-phenotype in suc2A9 cells because the fu- sion would not travel to the TGN. Thus, the invertase moiety could not be released by Kex2p, and subsequent movement to the cell surface would be prohibited. If, however, the fusion protein was capable of movement to the TGN, then Kex2p would release the invertase moiety, allowing the enzyme to travel to the cell surface resulting in a Suc ÷ phenotype. Therefore, the ability of various Ochlp-Kex2 site-invertase fusion proteins to confer a Suc ÷ phenotype to suc2A9 cells would be the first test of whether Ochlp is capable of movement to more distal Golgi compartments. A similar method has recently been used to study localization of Sec12p (8), a protein that cycles between the ER and Golgi, as well as Mnnlp (32), a glycosyltransferase localized to the trans-Golgi and TGN.
To detect Ochlp as well as the various fusion proteins by Western blot analysis and IP, we epitope tagged several of the proteins (Fig. 1) with the HA epitope recognized by the mAb, 12CA5 (110) . All proteins were expressed from single-copy centromere-based (CEN) plasmids under the control of the wild-type OCH1 promoter. All Ochlp epitope-tagged molecules and fusion proteins used in this study are capable of complementing the temperature-sensitive defect (65) of the ochl null strain (Table I) , suggesting that the proteins are properly folded and correctly localized. Aochl strains expressing the above constructs were tested for the ability to grow on rich media at 37°C. suc2A9 and suc2A9 Akex2 strains expressing the above constructs were tested for the ability to grow on yeast extract peptone-sucrose-containing media at 30°C. The Aochl strain is temperature sensitive, being unable to grow at 37°C.
Yeast strain SEY6210, which lacks a functional SUC2 gene and is therefore unable to utilize sucrose, was transformed with plasmids encoding the Ochlp-invertase fusion proteins diagrammed in Fig. 1 . The nomenclature used for portions of the various fusion proteins is as follows: O refers to Ochlp, H symbolizes the HA epitope, K indicates the Kex2p cleavage site, and I represents invertase. Only the Ochlp-invertase fusion proteins that contained an intervening Kex2p cleavage site, i.e., OKI and OHKI, but not OI, were able to restore the ability of the suc2A9 strain to use sucrose as a carbon source (Table I) . The ability to use sucrose was strictly dependent on the Kex2 protease because in an isogenic suc2A9 strain that lacks functional
Kex2p, OKI and OHKI were no longer able to functionally complement the suc2A9 mutation (Table I) . These data indicate that some Ochlp-invertase fusion protein is exposed to the Kex2p enzyme, resulting in invertase secretion. However, the level of invertase secretion does not need to be high to yield a Suc + phenotype, since as little as 1% of the wild-type level expressed from the SUC2 promoter suffices (46) .
To determine the amount of invertase released from OKI by Kex2p and subsequently secreted, the invertase activity of intact cells and cell lysates was quantitated (Table II). In the absence of the Kex2 cleavage site (OI), ~1% of the total cellular invertase was secreted. Cells expressing either OKI or OHKI secreted ~70% of the invertase produced. The high level of invertase secreted from cells expressing either OKI or OHKI suggests that the majority of fusion protein present in these cells at steady state is cleaved by Kex2p. To examine this finding further, we determined the amount of cleaved OHKI present in cells at steady state by immunoblot analysis using the anti-HA epitope antibody to detect OH and OHKI. Indeed, uncleaved OHKI, which migrates at ,~150 kD (see Fig. 3 ), is virtually undetectable in logarithmically growing cells (Fig. 2) . Instead, the only HA-immunoreactive protein in the strain expressing OHKI appears as a single band at ~66 kD that comigrates on SDS-PAGE with HA epitopetagged Oehlp. Only uncleaved OHKI is detected in a Akex2 strain (data not shown). Together, these results indicate that the large majority of the OHKI fusion protein is cleaved by the late Golgi Kex2 protease.
To determine the rate at which the OHKI fusion protein is cleaved by Kex2p, we performed a pulse-chase analysis. The yeast strain SEY6210 containing the pOHKI plasmid was labeled at 30°C for 2 min and chased for 20 min (Fig.  3) . Aliquots of cells were removed at 0, 0.5, 1, 2, 5, 10, and 20 min of chase. Labeled proteins were immunoprecipitated with the anti-HA epitope antibody, separated by SDS-PAGE, and visualized by autoradiography. At the 0-rain time point, the uncleaved OHKI fusion protein is evident migrating at ,'-~150 kD. The fusion is rapidly cleaved to yield HA epitope-tagged Ochlp (OH) and invertase, with a half-time of ~-,5 min. By 20 min, there is no detectable fusion protein remaining in the cell. Therefore, OHKI is rapidly (Fig. 3) and completely (Figs. 2 and 3 ) cleaved by the Kex2 protease. The invertase activity of strain SEY6210 expressing the above constructs was measured. Total activity from extracts prepared from lysed cells was determined by the method of Goldstein and Lampen (29) . Whole cell extracts were prepared to measure periplasmic activity (44) . Each value represents the mean of three experiments. The ~50% reduction in the total invertase activity in OKI and OHKI ceils relative to Ol cells is probably due to release of invertase into the media, which would have been undetected with the method used.
Although OHKI is not subject to the high degree of outerchain glycosylation as seen with wild-type invertase (102), a portion of the fusion protein migrates as a heterogeneous population of higher molecular mass polypeptides before cleavage by Kex2p (see the 5-min time point in Fig. 3 ). To examine whether this higher molecular mass "smear" represents fusion protein that had received outerchain glycosylation, we used mannose linkage-specific antibodies (Fig. 4) . Cells expressing OHKI were briefly pulse labeled as in Fig. 3 and then chased for 5 min before IP with anti-HA epitope antibody. The immunoprecipitated proteins were eluted from the anti-HA epitope antibody and subjected to a second IP with antibody specific for either the HA epitope, the oL-1,6-mannose linkage, which is added in the cis/medial Golgi, or the o~-l,3-mannose linkage, which is added in the trans/TGN compartments. The 150-kD OHKI fusion protein (Fig. 4 ) was immunoprecipitated by the a-l,6-mannose-specific antisera, indicating that it had obtained at least the "initial" o~-l,6-mannose residue in the cis-Golgi. A portion of the heterogeneous smear that migrates just slightly slower than the 150-kD protein could be immunoprecipitated by the a-l,6-mannose-specific antisera, but not the a-l,3-mannose-specific antisera (Fig. 4 , compare lanes 2 and 3). We believe this represents material that had progressed to the medial-Golgi, where the a-l,6 mannose outer-chain backbone was elongated, but had not moved to the trans-Golgi, and min. SEY6210 cells expressing OHKI were labeled with Tran35Slabel for 2 rain at 30°C and chased for the times indicated. HA epitope-tagged proteins from each time point were immunoprecipitated with the anti-HA epitope antibody, separated by SDS-PAGE, and visualized by autoradiography. The OHKI and OH (generated from OHKI) proteins are indicated on the left. therefore had not obtained terminal ot-l,3-mannose linkages. The higher molecular mass region of the smear was immunoprecipitable by both the a-l,6-and a-l,3-mannose-specific antibodies, indicating that this material had been exposed to the a-l,3 ManT in the trans-Golgi. Finally, as indicated previously, OH is derived from OHKI by Kex2p cleavage in the TGN. This experiment indicated that within 5 min of synthesis, OHKI receives all the modifications indicative of transit through the cis, medial, trans, and TGN compartments. Some OH derived from OHKI is also immunoprecipitable by the a-l,6-and the ot-l,3-mannose-specific antibodies, but to a lesser extent than the invertase-containing fusion protein. This may reflect the fact that Ochlp has only four potential glycosylation sites (65) compared to the 13 sites of invertase (13) . Another possibility is that the oligosaccharides on the Ochlp moiety are less heavily modified than those on the invertase moiety and therefore less immunoreactive (6) . The experiments presented above indicate that OHKI rapidly receives outer-chain glycosyl modifications and is cleaved by Kex2p, but do not clearly show where this cleavage is occurring. Three possibilities have been considered: (a) OHKI is cleaved by Kex2p in the ER or in transport vesicles bound for the Golgi, (b) OHKI is statically retained in the cis-Golgi and is cleaved in situ by newly synthesized Kex2p or Kex2p that may be recycling from the TGN (if this, in fact, occurs), and (c) OHKI travels to the TGN where it encounters Kex2p and is cleaved.
If Ochlp is cleaved by Kex2p in the TGN, then the fusion protein should obtain outer-chain glycosylation in the medialand trans-Golgi before cleavage. This appears to be the case in Fig. 3 , but because the processing is so fast relative to the pulse time, the two modifications may be occurring simultaneously to different subpopulations of labeled molecules, making clear kinetic resolution of the processes difficult. To circumvent this difficulty, we have used a secl8-1 temperature-sensitive strain, where transport of vesicles from the ER to the Golgi, and within the Golgi, is blocked at the restrictive temperature (31, 71) .
Isogenic SEC18 and sec18-1 strains expressing OHKI were labeled at 25°C for 7.5 min. Cells were chased at the restrictive temperature (37°C) for 0, 1, 2, and 5 min, and radiolabeled protein was immunoprecipitated with anti-HA epitope antibody. The antigens were then reimmunoprecipitated with the linkage-specific antibodies to determine how far the proteins had progressed through the secretory Figure 4 . The oligosaccharides of OHKI receive ct-1,6-and ct-l,3mannose linkages. SEY6210 cells expressing OHKI were labeled with Tran3SS-label for 2 min at 30°C and chased for 5 min at 30°C. Protein was immunoprecipitated with anti-HA epitope antibody. The immunoprecipitated protein was dissociated from the immune complex and divided into equivalent aliquots. The protein was then subjected to a second immunoprecipitation with either the anti-HA epitope antibody or with ct-l,6-or ct-l,3-mannose linkage-specific antisera. The precipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The OHKI and OH (generated from OHKI) proteins are indicated on the left. Our interpretation of how far each protein has progressed through the secretory pathway (see text) is indicated on the right. pathway before the block in transport. In the wild-type (SEC18) strain (Fig. 5, top panel) , OHKI obtained outerchain glycosylation, and the Golgi-modified fusion protein was rapidly cleaved to OH. During the chase period the levels of the c~-1,6-and c~-l,3-modified OHKI diminished, with a concomitant increase in the level of OH. Thus, a-l,6-and ct-l,3-modified OHKI is the precursor of OH.
The same experiment, done in a sec18-1 strain, revealed that less fusion protein had progressed to the Kex2p compartment in the sec18-1 strain than in the wild-type strain at the earliest timepoint (compare the levels of OH derived from OHKI at the 0-min time points). Therefore, the kinetics of vesicular transport in sec18-1 cells is slightly slower than in wild-type cells, even at the "permissive" temperature of 25°C. This probably reflects a reduction in the activity of Secl8p. Despite this kinetic effect, it is clear that in sec18-1 cells (Fig. 5, bottom panel) , conversion of OHKI to OH ceases after blocking vesicular transport at the restrictive temperature. This result is inconsistent with the first two possibilities described above. OHKI is not processed in the ER, in ER-derived transport vesicles, or in the cis-Golgi either by newly synthesized Kex2p or by Kex2p recycling back from the TGN. If this were occurring, the Kex2p present in the OHKI-bearing compartments would be trapped there after the temperature shift and continue to be active. The result would be generation of OH from OHKI, which is not observed.
This leaves the third possibility mentioned above: that OHKI is processed in the TGN. In support of this conclusion is the finding that neither the c~-1,6-nor ot-l,3-mannose-modified OHKI is converted to OH after imposition of the transport block (Fig. 5, bottom panel) in sec18-1 Figure 5 . Cleavage of OHKI occurs in the TGN. Isogenic SEC18 and secl8-1 strains (SH104 with or without pSEC18) expressing OHKI were labeled with Tran35S-label for 7.5 min at 25°C. At the 0-min time point, cells were shifted to 37°C and chased for the times indicated. Radiolabeled, HA epitope-tagged protein was immunoprecipitated with the anti-HA epitope antibody. The immune complexes were dissociated, and the protein was divided into equivalent aliquots and subjected to a second immunoprecipitation with either anti-HA epitope antibody or the ct-l,6-or ct-l,3-mannose linkage-specific antibodies. The proteins were separated by SDS-PAGE and visualized by autoradiography. The migration of the OHKI fusion protein and the cleaved OH are indicated on the left. cells. In fact, in sec18-1 cells, the ct-l,3-mannosylated fusion protein was not cleaved by Kex2p even after 1 h at 37°C (data not shown). This result indicates that processing by Kex2p only occurs after modification in the medialand trans-Golgi. Furthermore, the processing must occur in a separate compartment, distinct from the medialand trans-Golgi. Taken together, this analysis indicated that conversion of OHKI to OH does not take place in the ER, or the cis-, medial-, or trans-Golgi. We conclude that the processing of OHKI to OH occurs in the Kex2p compartment, the TGN.
To examine whether transit of the OHKI fusion protein to the TGN is reflective of the traffic pattern of wild-type Ochlp or whether it was induced by the presence of the in-vertase moiety in the fusion protein, we created a reporter molecule that more closely resembles wild-type Ochlp. This construct, termed O K H (Fig. 1) , consists of Ochlp followed by the Kex2 cleavage site and the H A epitope tag, but lacks the invertase moiety. Cleavage of the O K H protein by Kex2p will result in the removal of the H A epitope causing the protein to be refractory to IP by the anti-HA epitope antibody. O K H was expressed in isogenic suc2A9 and suc2zl9 Akex2 strains, and pulse-chase experiments were performed to follow the transit of the O K H protein (Fig. 6) . In the absence of Kex2p activity, the O K H is immunoprecipitable with the anti-HA epitope antibody for an extended period of time. However, in the presence of functional Kex2p, the O K H protein is rapidly cleaved by the protease with a half-time (~5 min) very similar to that seen for the O H K I fusion protein (Fig. 3) . Thus, movement of the fusion protein to the late Golgi is not induced by the invertase moiety.
To test directly whether H A epitope-tagged Ochlp (OH) is exposed to late Golgi compartments, cells expressing OH were labeled briefly, chased for 30 rain, and immunoprecipitated with the anti-HA epitope antibody followed by IP with the linkage-specific antibodies (Fig.  7) . We found that de novo-synthesized O H (i.e., not generated by cleavage of OHKI) was immunoprecipitable with the et-l,6-mannose-specific antisera and, to a lesser degree, with et-l,3-mannose-specific antisera. Although only a small amount of OH is immunoprecipitable with the a-l,3-mannose-specific antisera, this amount is similar to the amount of a-l,3-mannose-bearing O H generated from OHKI, which is known to have reached the TGN (Fig. 4) . The presence of comparable levels of o~-l,3-Man linkages on de novo-synthesized OH and OH generated from OHKI, suggests that at least some of the de novosynthesized OH has been exposed to the et-l,3-ManT, which is resident in both the trans-Golgi and the TGN (33).
These results (Figs. 6 and 7) indicate that O K H and OH undergo a similar traffic pattern as OHKI, and by extrapolation, we suggest that wild-type Ochlp may do so as well.
Figure Z De novo-synthesized OH bears a-l,6-, and to a lesser degree, a-l,3-mannose linkages. SEY6210 cells expressing OH were labeled for 5 rain at 30°C and chased for 30 min. Protein was immunoprecipitated with the anti-HA epitope antibody. The immune complex was dissociated by heating, and the precipitated protein was divided into equivalent aliquots. The protein was then subjected to a second immunoprecipitation with either the anti-HA epitope antibody or the ct-l,6-or c~-l,3-mannose linkage-specific antibodies. The proteins were separated by SDS-PAGE and visualized by autoradiography.
The experiments presented so far indicate that O H and O H K I rapidly travel to the TGN. To determine the fate of Ochlp after encountering the late Golgi, we first examined the stability of both de novo-synthesized O H and O H derived from OHKI (Fig. 8) . Cells were labeled for 10 rain at 30°C, chased for 180 min, and aliquots of cells were removed at various time points. O H (Fig. 8 A) and OH derived from O H K I (Fig. 8 B) have similar half-lives of ~60 min. This half-life is an order of magnitude longer than the half-time of transit of O H K I (Fig. 3) or OKH (Fig. 6) to the TGN. OH is stable in a Apep4 strain (data not shown)
indicating that the eventual degradation of Ochlp occurs in the vacuole (38) . These findings indicate that Ochlp is Figure 6 . Rapid transit to the TGN is not dependent on the invertase moiety. Isogenic KEX2 and Akex2 strains expressing OKH were labeled with Tran35S-label for 2 min at 30°C and chased for the times indicated. The proteins were immunoprecipitated with the anti-HA epitope antibody, separated by SDS-PAGE, and visualized by autoradiography. Figure 8 . De novo-synthesized OH and OH derived from OHKI persist in the cell 12-fold longer than the time taken to reach the TGN. SEY6210 cells expressing either OH or OHKI were labeled with Tran35S-label for 10 min at 30°C and chased for the times indicated. The proteins were immunoprecipitated with the anti-HA epitope antibody, separated by SDS-PAGE, and visualized by autoradiography. The migration of OH and OHKI are indicated.
not rapidly transported to the vacuole after reaching the late Golgi.
To confirm these results, we compared the subcellular fractionation behavior of an IMP of the vacuole, the 100-kD subunit of the H+-ATPase (21) (Fig. 9 A) , to that of OH, either synthesized de novo (Fig. 9 B) or generated from O H K I (Fig. 9 C) . These strains also expressed H A epitope-tagged Kex2p that will be discussed below. A lysate ($1) was prepared from strains expressing both H A epitope-tagged Kex2p and OH, or both H A epitopetagged Kex2p and OHKI. The $1 fractions were separated by differential centrifugation at 10,000 g into pellets (P10) Figure 9 . The compartment(s) bearing de novo-synthesized OH and OH derived from OHKI have similar buoyant densities, distinct from that of the vacuole and the Kex2-compartment. Lysates (S1) were prepared from zakex2 strains expressing HA epitope-tagged Kex2p and either (A and B) OH or (A and C) OHKI and separated by differential centrifugation into a pellet (PIO) and a Golgi-enriched supernatant ($10). A portion of each S10 was centrifuged to equilibrium in 2540% (wt/wt) sucrose gradients. The gradients were fractionated, and (D) the sucrose concentration of each gradient fraction was determined by refractometry. (A) The proteins in 2 ~1 of the S1, P10, and S10 were separated by 8% SDS-PAGE and analyzed by immunoblotting with the anti-yeast vacuolar H+-ATPase 100-kD subunit at a 1:4,000 dilution. (B and C) The proteins in 2 ~1 of the S1, P10, and S10, as well as 15 I~1 of each gradient fraction, were separated by 8% SDS-PAGE and analyzed by immunoblotting with the anti-HA epitope antibody at a 1:1,000 dilution. Protein was visualized using the chemiluminescence detection system. and supernatants (S10). The S1, P10, and S10 fractions were then probed by immunoblotting with an antibody specific for the 100-kD subunit of the H+-ATPase (21) and with the anti-HA epitope antibody to detect OH and H A epitope-tagged Kex2p. While the large majority of the H A epitope-tagged Ochlp and Kex2p fractionate into the 10,000-g supernatant (S10) (Fig. 9, B and C; left) , the vacuolar marker was exclusively found in the 10,000-g pellets (P10) (Fig. 9 A) . This again indicates that after OH encounters the TGN it does not reside in the vacuole.
To examine whether OH simply remains in the TGN after transit there, or whether it returns to the cis-Golgi, the Golgi-enriched $10 supernatants were fractionated by isopycnic centrifugation on 25-40% sucrose gradients ( Fig. 9, B-D) , and the distribution of OH and H A epitopetagged Kex2p, which is a marker of the TGN, was examined by immunoblotting. In both gradients Ochlp migrated in a peak (predominantly fractions 6-9) corresponding to ~28-32% (wt/wt) sucrose, whereas a more defined peak of Kex2p (predominantly fractions [4] [5] was evident at about 34% sucrose (Fig. 9, B-D) . Thus, at steady state, the bulk of OH, irrespective of whether it is synthesized de novo or derived from OHKI, resides in a compartment physically separable from both the Kex2p compartment and the vacuole.
To determine if the compartment that contained O H at steady state was indeed the cis-Golgi, we localized wildtype Ochlp. We used an antisera directed against the luminal domain of Ochlp for this purpose. When used to immunoprecipitate protein from whole cell extracts (Fig. 10  A) , the antibody recognizes a protein with an apparent molecular mass of 66 kD in the wild-type strain that is absent in the Aochl cells. To localize Ochlp, wild-type cells were labeled for 2 min at 30°C and chased for 60 min. A Golgi-enriched $10 supernatant was prepared from labeled cells and was fractionated on a 25-40% sucrose gradient. Protein from each gradient fraction was immunoprecipitated with the anti-Ochlp antisera and separated by SDS-PAGE (Fig. 10 B) . Ochlp was found in fractions 7-10 corresponding to 28-32% sucrose. Thus, the wildtype Ochlp compartment migrates at the same density observed for the compartment bearing H A epitope-tagged Ochlp, suggesting that the epitope-tagged protein returns to the cis-Golgi after encountering the TGN.
In another approach to demonstrate that the Ochlp fusion proteins reside in the cis-Golgi at steady state, we examined whether cells expressing only an Ochlp construct that rapidly encounters the Kex2p compartment can glycosylate invertase to the same degree as wild-type cells. Invertase provides a sensitive measure of the outer-chain glycosylation because it is hyper-glycosylated as it passes through the Golgi (24). Since Ochlp is responsible for initiation of this extensive glycosylation (64, 80) , a significant decrease in Ochlp activity should be evident as reduced glycosylation of invertase. Therefore, various strains (wildtype, Aochl , and Aochl expressing wild-type Ochlp [O], OH, or OKH) were labeled, and the glycosylation state of endogenous invertase was examined by immunoprecipitation and SDS-PAGE (Fig. 11 ). As expected, invertase is hyper-glycosylated in the wild-type strain, migrating as a heterogeneous smear. In contrast, invertase in the Aochl strain migrates as a distinct band because outer-chain glycosylation cannot be initiated. Expression of either wild-type Ochlp (O) or H A epitope-tagged Ochlp (OH) in the Aochl strain results in wild-type glycosylation of invertase. Significantly, expression of OKH, which encounters the Kex2p compartment with a half-time of 5 min (Fig. 6) , also results in hyper-glycosylation of invertase, suggesting that an Ochlp moiety known to transit to the late Golgi is fully active. Taken together, these data suggest a model in which the steady-state distribution of epitope-tagged Ochlp is in the cis-Golgi, yet the protein undergoes rapid movement through later compartments and is retrieved.
In an effort to determine if the behavior of H A epitopetagged Ochlp is representative of the wild-type protein, we examined the stability of wild-type Ochlp and compared it to OH (Fig. 12) . Wild-type cells expressing OH were labeled for 10 min at 30°C, chased for 240 min, and aliquots of cells were analyzed by IP with the anti-Ochlp antisera followed by SDS-PAGE. The anti-Ochlp antisera recognizes both wild-type Ochlp and OH, which migrates more slowly through the gel due to the presence of the H A epitope tag. The half-life of the wild-type protein is ~2.5fold longer than the tagged protein. Although the H A epitope tag does not seem to perturb the steady-state distribution of the protein (compare Figs. 9 and 10) , it may affect the kinetics of recycling, which will be discussed below.
Most recent models for the retention of type II IMPs in the Golgi suggest that these proteins cease their forward movement when they reach the appropriate compartment. It has been suggested that this process could be mediated by entry into a large oligomer, interaction with a putative Golgi matrix, or by virtue of a cholesterol-mediated gradient of membrane thickness across the Golgi stack (see Introduction). In contrast to these models we have found that the cis-Golgi protein used in our analyses can move past the cis compartment and subsequently be retrieved.
The yeast Golgi apparatus can be biochemically subdivided into at least four compartments (see Introduction). By analogy to mammalian Golgi, we refer to these as cis, Figure 12 . Wild-type Ochlp has a slightly longer half-life than HA epitope-tagged Ochlp. Wild-type (SEY6210) cells expressing the HA epitope-tagged Ochlp construct (pOH) were labeled with Tran35S-label for 10 min at 30°C and chased for the times indicated. Protein was immunoprecipitated with the anti-Ochlp antibody, separated by SDS-PAGE, and visualized by autoradiography. The migration of Ochlp and HA_ epitope-tagged Ochlp is indicated. The amount of Ochlp present at each time point was quantitated by Phosphorimager (Molecular Dynamics, Sunnyvale, CA) analysis. medial, trans, and TGN. The ManT, Ochlp, is thought to be a resident of the cis-Golgi in yeast. At the distal end of the Golgi is the TGN compartment that houses the Kex2p endoprotease responsible for the maturation of pro--a-factor and pro-killer toxin in S. cerevisiae.
To study Golgi localization in yeast, we have analyzed the traffic pattern of a series of Ochlp-invertase fusion proteins. The most instructive protein consisted of fulllength Ochlp, an HA epitope tag, a Kex2p cleavage site, and full-length invertase (OHKI). Surprisingly, we have found that OHKI is not efficiently retained in the cis-Golgi. This protein moves to the TGN, where it is processed by Kex2p, resulting in invertase secretion. Remarkably, this cleavage event occurs with a half-time of ~5 min, and is so efficient that the uncleaved fusion protein is undetectable at steady state. The oligosaccharides of the fusion protein receive medial (elongated ct-l,6-mannose) and trans (terminal o~-l,3-mannose) modifications before Kex2p cleavage. This indicates that the cleavage of OHKI occurs after the fusion protein has encountered the c/s-, medial-, and trans-Golgi. Newly synthesized Kex2p in transit through the cis compartment, or Kex2p recycling through the early Golgi, are not responsible for cleavage of OHKI. Thus, processing of OHKI occurs in the TGN.
OH is ultimately degraded in the vacuole in a PEP4dependent fashion. We found that the half-life of OH degradation, irrespective of whether it is synthesized at the ER or generated in the TGN, is 60 rain. This turnover time is 12-fold longer than the time required to reach the TGN, indicating that the OH does not move directly from the TGN to the vacuole, a result that was confirmed by subcellular fractionation. We have also shown that the bulk of both ER-synthesized OH and TGN-generated OH resides in a compartment with the same density as wild-type Ochlp and a lower buoyant density than the TGN. This indicates that OH derived from OHKI in the TGN is correctly localized to the cis-Golgi at steady state despite its rapid transit through the TGN. Finally, we found that cells exclusively expressing an Ochlp construct that rapidly moves through the TGN can glycosylate invertase as efficiently as wild-type cells. Taken together, these findings indicate that the cis-Golgi protein Ochlp is capable of recycling from the TGN.
The rapid transit time (~5 min) to the TGN, although slower than the ~1.5 min required for invertase to acquire outer-chain oligosaccharide modification (86) , is much too fast to represent efficient retention in the cis-Golgi. Nevertheless, the finding that OH recycles does not rule out the possibility that there is a "statically retained" state. It does imply, however, that entry into this putative retained state is not an efficient process. In fact, it is difficult to imagine a model that allows for distinct protein compositions in different cisternae that does not involve at least a transiently retained state. One can view the process of retention as a pause in a particular compartment. At one extreme, the protein "pauses" for its entire lifetime (that is, it has a high affinity for the retained state), after which it is released and degraded. In the other extreme, the pause is short compared to the lifetime of the protein (a low affinity for the retained state), and after continued movement the protein can be retrieved from distal compartments. This recycling continues until the protein finally escapes the retrieval system and is degraded. The degree to which differ-ent proteins pause may vary. In this kinetic retention model epitope-tagged Ochlp would pause only briefly, possibly because of a low affinity for the putative retained state, but this effect would be sufficient to result in the predominantly c/s-Golgi distribution of OH at steady state.
These findings are in accordance with a recent study of the localization of Mnnlp (32) , which is the terminal tx-l,3-ManT in yeast. Mnnlp is distinctive because of its approximately equal distribution between two physically separable compartments, one of which contains Kex2p (33) ; that is, Mnnlp resides in both the trans-Golgi and TGN compartments. Recently, Graham and Krasnov (32) used a fusion protein consisting of the Mnnlp transmembrane domain followed by a Kex2p site and invertase to examine the extent of exposure of this protein to the Kex2p enzyme. They found that 85% of the protein was rapidly processed by Kex2p, which is more than could be accounted for by the dual steady-state distribution of Mnnlp in the trans-Golgi and TGN. Therefore, they suggested that Mnnlp may cycle between the trans-Golgi and TGN. In • the same study, however, it was also shown that the luminal domain of Mnnlp, which was not included in the fusion protein, also contains localization information. Accordingly, the authors pointed out that the disproportionately high exposure of the fusion protein to the TGN might be due to the absence of the Mnnlp luminal domain. Our results with full-length Ochlp constructs tend to support Graham and Krasnov's suggestion that Mnnlp cycles between the trans and TGN compartments.
Another outcome of our analyses with Ochlp is the clear demonstration of a retrograde transport pathway from the TGN to the cis-Golgi. This extends the recent demonstration by Schrrder et al. (88) that a type I medial-Golgi protein, Emp47, has a recycling itinerary that extends from the trans-Golgi (i.e., the et-l,3-ManT compartment) to the ER. There are also several precedents for retrograde transport through the Golgi apparatus in mammalian cells. First, several residents of the secretory pathway have been shown to obtain oligosaccharide modifications that occur in later compartments. For example, the oligosaccharides of the ER resident calreticulin are terminally galactosylated, a modification that occurs in the trans-Golgi (76) . Likewise, two medial-Golgi resident proteins, GIMPc (112) and MG160 (30) , have been shown to bear terminal sialic acid residues, which are added in the TGN. In addition, the N-acetylglucosaminyl-transferase I enzyme, a resident of the medial/trans-Golgi, can undergo retrograde transport to the early Golgi (40) . Second, several toxins which are taken up by endocytosis, such as Pseudomonas exotoxin, ultimately gain access to the cytosol via the ER (73, 75) and their cytotoxicity can be increased by placing a KDEL sequence at their carboxy termini (93) . And finally, it was recently shown that a short peptide that contains KDEL at the carboxy terminus can move via retrograde transport from the TGN to the ER (60) .
Although an epitope-tagged version of Ochlp has been used for many of the experiments presented, a series of experiments has been performed to determine if the traffic pattern of the fusion protein is representative of the wildtype protein. One could envision that the bulky invertase moiety appended to the carboxy terminus of Ochlp could somehow interfere with retention, perhaps by inhibiting entry into a putative oligomer. We have shown that is not the case because a construct without the invertase moiety encounters the TGN with the same kinetics as the invertase-bearing fusion. In addition, we have shown that Ochlp, altered only by the addition of an HA epitope tag (OH), obtained the a-l,6-and ot-l,3-mannose linkages characteristic of transit through the medial and trans compartments. This indicates that neither the specific sequences present in the Kex2 cleavage site nor invertase are responsible for continued forward movement. Finally, Ochlp--invertase fusion protein-dependent secretion of invertase is not dependent on the HA epitope tag (Tables  I and II) . Thus, movement to distal Golgi compartments is not dependent on any of the specific sequences appended to the carboxy terminus of Ochlp. However, it is possible that modification of the carboxy terminus itself, independent of the protein sequence (HA epitope, Kex2 site, or invertase), is responsible for this traffic pattern.
Several experiments suggest that epitope-tagged Ochlp behaves similarly to the native protein. First, epitopetagged Ochlp appears to be localized to the same compartment as wild-type Ochlp at steady state (Figs. 9 and 10). In addition, the fusion constructs appear to be fully active, as measured by their ability to glycosylate invertase, again suggesting that the proteins are correctly localized at steady state. However, it is important to note that the half-life of wild-type Ochlp is about 2.5-fold longer than the half-life of epitope-tagged Ochlp. There are several possible reasons for this difference. One is that the wild-type protein may enter into the putative retained state with greater efficiency than the epitope-tagged protein. The majority of the Ochlp would enjoy a lengthy retained state, and only a small percentage of the Ochlp would travel to the late Golgi where it could be either retrieved by retrograde transport or shunted to the vacuole for degradation. If this is true, then the rapid flux demonstrated by the tagged molecule OKH, for example, could indicate that a retention signal exists at the carboxy terminus of Ochlp that is disrupted by the addition of the HA epitope. Alternatively, both wild-type and epitope-tagged Ochlp may enter into the putative retained state with a similar efficiency, but retrieval from the TGN may be compromised by the presence of the epitope tag, resulting in diversion to the vacuole.
Nevertheless, despite the kinetic difference between OH and wild-type Ochlp, it is now apparent that the localization mechanism of Ochlp involves a retrograde transport component. The majority of epitope-tagged Ochlp is transported to the late Golgi and must be recycled back to the cis-Golgi using a retrograde transport system. It is likely that the wild-type protein uses the same transport system, at least to some extent. It remains to be determined whether recycling plays a major or supporting role in the localization of the wild-type protein.