Development of a homogeneous MAP kinase reporter gene screen for the identification of agonists and antagonists at the CXCR1 chemokine receptor.

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the Galpha(i/o) or Galpha(q) families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sap1a following MAP kinase activation. In contrast to other reporter gene assays for Galpha(i/o)-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The Galpha(i)-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC(50) = 7.0 +/- 0.1) > GCP-2 (pEC(50) = 6.3 +/- 0.1) > NAP-2 (pEC(50) < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive Galpha(i/o)-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.

Cvt19 is a receptor for the cytoplasm-to-vacuole targeting pathway.

Cvt19 is specifically required for the transport of resident vacuolar hydrolases that utilize the cytoplasm-to-vacuole targeting (Cvt) pathway. Autophagy (Apg) and pexophagy, processes that use the majority of the same protein components as the Cvt pathway, do not require Cvt19. Cvt19GFP is localized to punctate structures on or near the vacuole surface. Cvt19 is a peripheral membrane protein that binds to the precursor form of the Cvt cargo protein aminopeptidase I (prAPI) and travels to the vacuole with prAPI. These results suggest that Cvt19 is a receptor protein for prAPI that allows for the selective transport of this protein by both the Cvt and Apg pathways.

Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole.

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase.

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.

Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting.

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy. In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p. Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated. Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions.

Alternative protein sorting pathways.

The term "nonclassical protein targeting" has been used to describe those pathways that have been recently discovered and differ mechanistically from the more studied "classical pathways." Because this nomenclature is rather arbitrary in terms of cellular relevance, we have chosen to group these protein sorting mechanisms under the heading "alternative protein sorting pathways" for the purpose of this review. Many of the alternative targeting pathways described are of primary importance. For example, without retrograde transport, both membrane material and targeting machinery accumulate at distal sites in the endomembrane system, preventing anterograde transport. Further, lysosome/vacuole delivery of degradative substrates by autophagic pathways is central to the role of this organelle as a primary site for intracellular degradation. Finally, targeting through the classical CPY pathway requires the ALP pathway for delivery of the vacuolar t-SNARE Vam3p. Analysis of these alternative targeting pathways provides a more complete understanding of eukaryotic cellular physiology.

Apg5p functions in the sequestration step in the cytoplasm-to-vacuole targeting and macroautophagy pathways.

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5(ts). Biochemical analysis of API import in the apg5(ts) strain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in the apg5(ts) mutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.

The itinerary of a vesicle component, Aut7p/Cvt5p, terminates in the yeast vacuole via the autophagy/Cvt pathways.

Aminopeptidase I (API) is delivered to the yeast vacuole by one of two alternative pathways, cytoplasm to vacuole targeting (Cvt) or autophagy, depending on nutrient conditions. Genetic, morphological, and biochemical studies indicate that the two pathways share many of the same molecular components. The Cvt pathway functions during vegetative growth, while autophagy is induced during starvation. Both pathways involve the formation of cytosolic vesicles that fuse with the vacuole. In either case, the mechanism of vesicle formation is not known. Autophagic uptake displays a greater capacity for cytosolic protein sequestration. This suggests the involvement of an inducible protein(s) that allows the vesicle-forming machinery to adapt to the increased degradative needs of the cell. We have analyzed the biosynthesis of Aut7p, a protein required for both pathways. We find Aut7p expression is induced by nitrogen starvation. Aut7p is degraded by a process dependent on both proteinase A and Cvt/autophagy components. Protease accessibility assays demonstrate that Aut7p is located within vesicles in strains defective in vesicle delivery or breakdown. Finally, the aut7/cvt5 mutant accumulates precursor API at a stage prior to vesicle completion. These data suggest that Aut7p is induced during autophagy and delivered to the vacuole together with precursor API by Cvt/autophagic vesicles.

Apg7p/Cvt2p is required for the cytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways.

Proper functioning of organelles necessitates efficient protein targeting to the appropriate subcellular locations. For example, degradation in the fungal vacuole relies on an array of targeting mechanisms for both resident hydrolases and their substrates. The particular processes that are used vary depending on the available nutrients. Under starvation conditions, macroautophagy is the primary method by which bulk cytosol is sequestered into autophagic vesicles (autophagosomes) destined for this organelle. Molecular genetic, morphological, and biochemical evidence indicates that macroautophagy shares much of the same cellular machinery as a biosynthetic pathway for the delivery of the vacuolar hydrolase, aminopeptidase I, via the cytoplasm-to-vacuole targeting (Cvt) pathway. The machinery required in both pathways includes a novel protein modification system involving the conjugation of two autophagy proteins, Apg12p and Apg5p. The conjugation reaction was demonstrated to be dependent on Apg7p, which shares homology with the E1 family of ubiquitin-activating enzymes. In this study, we demonstrate that Apg7p functions at the sequestration step in the formation of Cvt vesicles and autophagosomes. The subcellular localization of Apg7p fused to green fluorescent protein (GFP) indicates that a subpopulation of Apg7pGFP becomes membrane associated in an Apg12p-dependent manner. Subcellular fractionation experiments also indicate that a portion of the Apg7p pool is pelletable under starvation conditions. Finally, we demonstrate that the Pichia pastoris homologue Gsa7p that is required for peroxisome degradation is functionally similar to Apg7p, indicating that this novel conjugation system may represent a general nonclassical targeting mechanism that is conserved across species.

Delivery of proteins and organelles to the vacuole from the cytoplasm.

The vacuole/lysosome is a primary catabolic site in the eukaryotic cell. One implication of its cellular role is that delivery systems must exist to target both hydrolytic enzymes and substrates destined for degradation to this organelle. A number of nonclassical vacuolar targeting pathways that deliver degradative substrates and at least one resident enzyme from the cytosol to the vacuole have recently been described. The pathways identified so far include cytoplasm to vacuole targeting, macroautophagy, pexophagy and vacuolar import and degradation. Cytological, genetic and molecular genetic approaches have begun to provide insight into the molecular basis of these processes.

Aminopeptidase I is targeted to the vacuole by a nonclassical vesicular mechanism.

The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.

Transport of a large oligomeric protein by the cytoplasm to vacuole protein targeting pathway.

Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.

Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome.

Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plant, and fungal cells that allows for both recycling of macromolecular constituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the molecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this pathway in yeast. Using the vacuolar hydrolase aminopeptidase I (API) as a marker, we provide evidence that the autophagic pathway overlaps with the biosynthetic pathway, cytoplasm to vacuole targeting (Cvt), used for API import. Before targeting, the precursor form of API is localized mostly in restricted regions of the cytosol as a complex with spherical particles (termed Cvt complex). During vegetative growth, the Cvt complex is selectively wrapped by a membrane sac forming a double membrane-bound structure of approximately 150 nm diam, which then fuses with the vacuolar membrane. This process is topologically the same as macroautophagy induced under starvation conditions in yeast (Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994. J. Cell Biol. 124:903-913). However, in contrast with autophagy, API import proceeds constitutively in growing conditions. This is the first demonstration of the use of an autophagy-like mechanism for biosynthetic delivery of a vacuolar hydrolase. Another important finding is that when cells are subjected to starvation conditions, the Cvt complex is now taken up by an autophagosome that is much larger and contains other cytosolic components; depending on environmental conditions, the cell uses an alternate pathway to sequester the Cvt complex and selectively deliver API to the vacuole. Together these results indicate that two related but distinct autophagy-like processes are involved in both biogenesis of vacuolar resident proteins and sequestration of substrates to be degraded.

Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole.

The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway. Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants. In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole. Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy. Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway. However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.

Identification of a cytoplasm to vacuole targeting determinant in aminopeptidase I.

Aminopeptidase I (API) is a soluble leucine aminopeptidase resident in the yeast vacuole (Frey, J., and K.H. Rohm. 1978. Biochim. Biophys. Acta. 527:31-41). The precursor form of API contains an amino-terminal 45-amino acid propeptide, which is removed by proteinase B (PrB) upon entry into the vacuole. The propeptide of API lacks a consensus signal sequence and it has been demonstrated that vacuolar localization of API is independent of the secretory pathway (Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. J. Cell Biol. 119:287-299). The predicted secondary structure for the API propeptide is composed of an amphipathic alpha-helix followed by a beta-turn and another alpha-helix, forming a helix-turn-helix structure. With the use of mutational analysis, we determined that the API propeptide is essential for proper transport into the vacuole. Deletion of the entire propeptide from the API molecule resulted in accumulation of a mature-sized protein in the cytosol. A more detailed examination using random mutagenesis and a series of smaller deletions throughout the propeptide revealed that API localization is severely affected by alterations within the predicted first alpha-helix. In vitro studies indicate that mutations in this predicted helix prevent productive binding interactions from taking place. In contrast, vacuolar import is relatively insensitive to alterations in the second predicted helix of the propeptide. Examination of API folding revealed that mutations that affect entry into the vacuole did not affect the structure of API. These data indicate that the API propeptide serves as a vacuolar targeting determinant at a critical step along the cytoplasm to vacuole targeting pathway.

A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: energetics and mechanistic implications.

Chloroplast protein import presents a complex membrane traversal problem: precursor proteins must cross two envelope membranes to reach the stromal compartment. This work characterizes a new chloroplast protein import intermediate which has completely traversed the outer envelope membrane but has not yet reached the stroma. The existence of this intermediate demonstrates that distinct protein transport machineries are present in both envelope membranes, and that they are able to operate independently of one another under certain conditions. Energetic characterization of this pathway led to the identification of three independent energy-requiring steps: binding of the precursor to the outer envelope membrane, outer membrane transport, and inner membrane transport. Localization of the sites of energy utilization for each of these steps, as well as their respective nucleotide specificities, suggest that three different ATPases mediate chloroplast envelope transport.

In vitro reconstitution of cytoplasm to vacuole protein targeting in yeast.

Although the majority of known vacuolar proteins transit through the secretory pathway, two vacuole-resident proteins have been identified that reach this organelle by an alternate pathway. These polypeptides are targeted to the vacuole directly from the cytoplasm by a novel import mechanism. The best characterized protein that uses this pathway is aminopeptidase I (API). API is synthesized as a cytoplasmic precursor containing an amino-terminal propeptide that is cleaved off when the protein reaches the vacuole. To dissect the biochemistry of this pathway, we have reconstituted the targeting of API in vitro in a permeabilized cell system. Based on several criteria, the in vitro import assay faithfully reconstitutes the in vivo reaction. After incubation under import conditions, API is processed by a vacuolar-resident protease, copurifies with a vacuole-enriched fraction, and becomes inaccessible to the cytoplasm. These observations demonstrate that API has passed from the cytoplasm to the vacuole. The reconstituted import process is dependent on time, temperature, and energy. ATP gamma S inhibits this reaction, indicating that API transport is ATP driven. API import is also inhibited by GTP gamma S, suggesting that this process may be mediated by a GTP-binding protein. In addition, in vitro import requires a functional vacuolar ATPase; import is inhibited both in the presence of the specific V-ATPase inhibitor bafilomycin A1, and in a yeast strain in which one of the genes encoding a V-ATPase subunit has been disrupted.

Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway.

In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.

Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana.

Cyclophilin (CyP), a protein with peptidyl-prolyl cis-trans isomerase (rotamase) activity, is the specific cellular target of cyclosporin A. We have isolated cDNA clones of two genes (designated ROC1 and ROC4) encoding CyP homologs from Arabidopsis thaliana (L.). The protein products of these genes are distinct from a previously identified Arabidopsis CyP. ROC1 is expressed in all tested plant organs and encodes a protein which is highly similar to previously described cytosolic CyP isoforms of other plants. In contrast, ROC4 is expressed only in photosynthetic organs and encodes a protein which includes an amino-terminal extension with properties of known chloroplast transit peptides. In vitro import experiments using the putative precursor protein to ROC4 showed that the protein is imported into chloroplasts where it is processed to the predicted mature size. Rotamase assays and immunoblot analysis of subcellular fractions indicate the presence of a CyP isoform in the stroma of chloroplasts but not in the thylakoid membranes or thylakoid lumen. Together, these data show that ROC4 is a novel CyP isoform which is located in the stroma of chloroplasts. In vitro chloroplast import of precursors of other chloroplast proteins was unaffected by concentrations of cyclosporin A which completely inhibit rotamase activity of chloroplast stromal CyP. Thus, this activity is not essential for protein import into chloroplasts.

Protein import into chloroplasts.

The structural complexity of chloroplasts is reflected in their intriguing protein-targeting system. Not only must nucleus-encoded proteins be targeted to the chloroplast, but also, once inside the chloroplast, these polypeptides must be directed to their final destination in one of six intrachloroplastic compartments. Although the details of this process remain elusive, many recent advances have improved our vantage point for examining this system.