Cranio-lenticulo-sutural dysplasia associated with defects in collagen secretion.

Cranio-lenticulo-sutural dysplasia (CLSD) is a rare autosomal recessive syndrome manifesting with large and late-closing fontanels and calvarial hypomineralization, Y-shaped cataracts, skeletal defects, and hypertelorism and other facial dysmorphisms. The CLSD locus was mapped to chromosome 14q13-q21 and a homozygous SEC23A F382L missense mutation was identified in the original family. Skin fibroblasts from these patients exhibit features of a secretion defect with marked distension of the endoplasmic reticulum (ER), consistent with SEC23A function in protein export from the ER. We report an unrelated family where a male proband presented with clinical features of CLSD. A heterozygous missense M702V mutation in a highly conserved residue of SEC23A was inherited from the clinically unaffected father, but no maternal SEC23A mutation was identified. Cultured skin fibroblasts from this new patient showed a severe secretion defect of collagen and enlarged ER, confirming aberrant protein export from the ER. Milder collagen secretion defects and ER distention were present in paternal fibroblasts, indicating that an additional mutation(s) is present in the proband. Our data suggest that defective ER export is the cause of CLSD and genetic element(s) besides SEC23A may influence its presentation.

Genetic and biochemical analysis of vesicular traffic in yeast.

Secretory, vacuolar and membrane protein transport in yeast occurs by processes that are highly conserved in eukaryotic cells. Recent years have seen a proliferation of approaches to the study of vesicular traffic, and in certain instances key breakthroughs have been achieved through the application of genetic and biochemical methods that are well suited to yeast as an experimental organism. The availability of the genetic approach has led to molecular insights concerning the mechanisms of vesicle biogenesis, targeting and fusion.

COPII and secretory cargo capture into transport vesicles.

Yeast cylosolic coat proteins (COPII) direct the formation of vesicles from the endoplasmic reticulum. The vesicles selectively capture both cargo molecules and the secretory machinery that is necessary for the fusion of the vesicle with the recipient compartment, the Golgi apparatus. Recent efforts have aimed to understand how proteins are selected for inclusion into these vesicles. A variety of cargo adaptors may concentrate and sort secretory and membrane proteins by direct or indirect interaction with a subset of coat protein subunits.

ER export: public transportation by the COPII coach.

The COPII coat produces ER-derived transport vesicles. Recent findings suggest that the COPII coat is a highly dynamic polymer and that efficient capture of cargo molecules into COPII vesicles depends on several parameters, including export signals, membrane environment, metabolic control and the presence of a repertoire of COPII subunit homologues.

The role of coat proteins in the biosynthesis of secretory proteins.

The biosynthesis of secretory proteins requires vesicle-mediated transport between the organelles of the secretory pathway. Biochemical and genetic analysis of the secretory pathway has identified two non-clathrin coats--COPI and COPII--that drive the formation of vesicles that mediate transport between the endoplasmic reticulum and the Golgi apparatus, and through the compartments of the Golgi. Recently, a molecular description of the subunits of these coats and the development of biochemical reagents to study their function has yielded new information on how these proteins share the task of organizing vesicle traffic early in the secretory pathway.

Dynamics of the COPII coat with GTP and stable analogues.

We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time. We show that with Sar1pGTP bound to liposomes, a single round of assembly and disassembly of the COPII coat lasts a few seconds. The two large COPII complexes Sec23/24p and Sec13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes. This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p complex. Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p.

Traffic COPs and the formation of vesicle coats.

Forward and retrograde trafficking of secretory proteins between the endoplasmic reticulum and the Golgi apparatus is driven by two biochemically distinct vesicle coats, COPI and COPII. Assembly of the coats on their target membranes is thought to provide the driving force for membrane deformation and the selective packaging of cargo and targeting molecules into nascent transport vesicles. This review describes our current knowledge on these issues and discusses how the two coats may be differentially targeted and assembled to achieve protein sorting and transport within the early secretory pathway.

Out of the ER--outfitters, escorts and guides.

The endoplasmic reticulum (ER) contains a variety of specialized proteins that interact with secretory proteins and facilitate their uptake into transport vesicles destined for the Golgi apparatus. These accessory proteins might induce and/or stabilize a conformation that is required for secretion competence or they might be directly involved in the sorting and uptake of secretory proteins into Golgi-bound vesicles. Recent efforts have aimed to identify and characterize the role of several of these substrate-specific accessory proteins.

Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae.

To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1. Sanders et al. (Sanders, S. L., K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman. 1992. Cell. 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore. We now show that mutation of a conserved residue (A181-->T) (Nelson, M. K., T. Kurihara, and P. Silver. 1993. Genetics. 134:159-173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203. We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired. Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect. Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane.

A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome.

Reconstituted proteoliposomes derived from solubilized yeast microsomes are able to translocate a secreted yeast mating pheromone precursor (Brodsky, J. L., S. Hamamoto, D. Feldheim, and R. Schekman. 1993. J. Cell Biol. 120:95-107). Reconstituted proteoliposomes prepared from strains with mutations in the SEC63 or KAR2 genes are defective for translocation; the kar2 defect can be overcome by the addition of purified BiP (encoded by the KAR2 gene). We now show that addition of BiP to wild-type reconstituted vesicles increases their translocation efficiency three-fold. To identify other ER components that are required for translocation, we purified a microsomal membrane protein complex that contains Sec63p. We found that the complex also includes BiP, Sec66p (gp31.5), and Sec67p (p23). The Sec63p complex restores translocation activity to reconstituted vesicles that are prepared from a sec63-1 strain, or from cells in which the SEC66 or SEC67 genes are disrupted. BiP dissociates from the complex when the purification is performed in the presence of ATP gamma S or when the starting membranes are from yeast containing the sec63-1 mutation. We conclude that the purified Sec63p complex is active and required for protein translocation, and that the association of BiP with the complex may be regulated in vivo.

Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex.

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.

SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae.

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S-transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.

Export of major cell surface proteins is blocked in yeast secretory mutants.

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction. Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway. All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.

Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae.

Secretion of cell wall-bound acid phosphatase by Saccharomyces cerevisiae occurs along a restricted portion of the cell surface. Acid phosphatase activity produced during derepressed synthesis on a phosphate-limited growth medium is detected with an enzyme-specific stain and is localized initially to the bud portion of a dividing cell. After two to three generations of phosphate-limited growth, most of the cells can be stained; if further phosphatase synthesis is repressed by growth in excess phosphate, dividing cells are produced in which the parent but not the bud can be stained. Budding growth is interrupted in alpha-mating-type cells by a pheromone (alpha-factor) secreted by the opposite mating type; cell surface growth continues in the presence of alpha-factor and produces a characteristic cell tip. When acid phosphatase synthesis is initiated during alpha-factor treatment, only the cell tip can br stained; when phosphate synthesis is repressed during alpha-factor treatment, the cell body but not the tip can be stained. A mixture of derepressed alpha cells and phosphatase-negative alpha cells form zygotes in which mainly one parent cell surface can be stained. The cell cycle mutant, cdc 24 (Hartwell, L.H. 1971. Exp. Cell Res. 69:265-276), fails to bud and, instead, expands symmetrically as a sphere at a nonpermissive temperature (37 degrees C). This mutant does not form a cell tip during alpha-factor treatment at 37 degrees C, and although acid phosphatade secretion occurs at this temperature, it is not localized. These results suggest that secretion reflects a polar mode of yeast cell- surface growth, and that this organization requires the cdc 24 gene product.

Reconstitution of retrograde transport from the Golgi to the ER in vitro.

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369-377). Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.

Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes.

In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis-Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed.

Characteristics of endoplasmic reticulum-derived transport vesicles.

We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast.

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.