Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs

The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane

A carboxyl-terminal hydrophobic domain is an essential component of the processed signal for attachment of the glycosyl-phosphatidylinositol (GPI) membrane anchor to proteins and it is linked to the site (omega) of GPI modification by a spacer domain. This study was designed to test the hypothesis that the hydrophobic domain interacts with the lipid bilayer of the endoplasmic reticulum (ER) membrane to optimally position the omega site for GPI modification. The hydrophobic domain of the GPI signal in the human folate receptor (FR) type alpha was substituted with the carboxyl-terminal segment of the low-density lipoprotein receptor (LDLR), including its membrane spanning region, without altering either the spacer or the omega site. The FR-alpha/LDLR chimera was not GPI modified but was attached to the plasma membrane by a polypeptide anchor. When the carboxyl-terminal half of the hydrophobic transmembrane polypeptide in the FR-alpha/LDLR chimera was altered by introduction of negatively charged (Asp) residues, or when the cytosolic domain in the chimera was deleted, the mutated proteins became GPI-anchored. On the other hand, attachment of a carboxyl-terminal segment of LDLR including the entire cytosolic domain to FR-alpha converted it into a transmembrane protein. The results indicate that in the FR-alpha/LDLR chimera the inability of the cellular machinery for GPI modification to recognize the hydrophobic domain is not due to the intrinsic nature of the peptide, but is rather due to the retention of the peptide within the lipid bilayer. It follows that the hydrophobic domain in the signal for GPI modification must traverse the ER membrane prior to recognition of the omega site by the GPI-protein transamidase. The results thus establish a critical topographical requirement for recognition of the GPI signal in the ER.     

Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

The NHB1 (N-terminal homology box 1) sequence in transcription factor Nrf1 is required to anchor it to the endoplasmic reticulum and also to enable its asparagine-glycosylation

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is negatively controlled by its NTD (N-terminal domain) that lies between amino acids 1 and 124. This domain contains a leucine-rich sequence, called NHB1 (N-terminal homology box 1; residues 11-30), which tethers Nrf1 to the ER (endoplasmic reticulum). Electrophoresis resolved Nrf1 into two major bands of approx. 95 and 120 kDa. The 120-kDa Nrf1 form represents a glycosylated protein that was present exclusively in the ER and was converted into a substantially smaller polypeptide upon digestion with either peptide:N-glycosidase F or endoglycosidase H. By contrast, the 95-kDa Nrf1 form did not appear to be glycosylated and was present primarily in the nucleus. NHB1 and its adjacent residues conform to the classic tripartite signal peptide sequence, comprising n-, h- and c-regions. The h-region (residues 11-22), but neither the n-region (residues 1-10) nor the c-region (residues 23-30), is required to direct Nrf1 to the ER. Targeting Nrf1 to the ER is necessary to generate the 120-kDa glycosylated protein. The n-region and c-region are required for correct membrane orientation of Nrf1, as deletion of residues 2-10 or 23-30 greatly increased its association with the ER and the extent to which it was glycosylated. The NHB1 does not contain a signal peptidase cleavage site, indicating that it serves as an ER anchor sequence. Wild-type Nrf1 is glycosylated through its Asn/Ser/Thr-rich domain, between amino acids 296 and 403, and this modification was not observed in an Nrf1(Delta299-400) mutant. Glycosylation of Nrf1 was not necessary to retain it in the ER.     

Post-Translational Acquisition of Ligand Binding-and Tyrosine Kinase-Domain Function by the Epidermal Growth Factor and Insulin Receptors

Abstract

The epidermal growth factor receptor (EGFR) and insulin receptor undergo slow post-translational modification by which they acquire hormone binding and tyrosine kinase (EGFR) function. The half-time for acquisition of EGF or insulin binding activity is 30-40 min and of tyrosine kinase activity (EGFR), is 10-15 min. Tunicamycin, an inhibitor of N-linked oligosaccharide addition, blocks acquisition of both EGF and insulin binding activity. With EGFR, activation precedes acquisition of resistance to endoglucos-aminidase H (t_1/2 75 min), a medial Golgi event. Treatment of active high mannose receptor with endo H generates fully active aglyco-receptor; thus, core oligosaccharide addition is a prerequisite for activation, but not for EGF binding per se. EGFR is activated in and translocated from the endoplasmic reticulum (ER) slowly (t_1/2 75 min). Since translocation rate equals the rate for acquisition of endo H resistance, transit from the ER is rate limiting for EGFR maturation. Tunicamycin inhibits exit from the ER parallel to its effect on acquisition of binding activity. Insulin proreceptor, a 210 kDa high-mannose glycopolypeptide, acquires insulin binding function (t_1/2 45 min) then is proteolytically cleaved (t_1/2 3 hr) into subunits of the mature α₂β₂ receptor. Modification giving rise to insulin binding activity is due to a conformational change in the binding domain, since human autoimmune antibody recognizes only the active species, while rabbit polyclonal antibody recognizes all forms. Newly-translated EGF proreceptor lacks a functional tyrosine domain capable of autophosphorylation; 30-40 min after translation, while still in     

GM1 Synthase Depends on N-Glycosylation for Enzyme Activity and Trafficking to the Golgi Complex

Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse 1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [³H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1–52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.     

Role of phosphorylation of the cytoplasmic domain of the alpha(2)-macroglobulin receptor (LRP)

The low density lipoprotein receptor-related protein (alpha(2)MR/LRP) is a cell surface receptor which is present on most cells and tissues. We show that the 85 kDa subunit, containing the transmembrane region and cytoplasmic domain is phosphorylated in vivo. Comparison of the phosphorylation of the low density lipoprotein receptor (LDLR) with a chimeric receptor containing the cytoplasmic domain of the alpha(2)MR/LRP (LDLR/LRP) showed that phosphorylation is exclusive to the cytoplasmic domain. Staurosporine, a general kinase inhibitor, resulted in a 40% lowering of phosphorylation of LDLR/LRP, but did not give rise to measurable changes in its membrane traffic in MDCK cells. The role of phosphorylation on degradation of the receptor was studied using inhibitors of lysosomal and proteasomal degradation. These studies showed that LDLR/LRP was rapidly turned over by proteasomal degradation but that this turnover was also not a consequence of phosphorylation.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible Degrader of the LDLR (IDOL)

We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease.     

Cell-to-cell transport via the lumen of the endoplasmic reticulum

Plasmodesmata are plasma membrane‐lined channels through which cytoplasmic molecules move from cell‐to‐cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3‐kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4‐kDa recombinant ER‐lumenal reporter protein (LRP) from a fragment of the endogenous ER‐lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER‐GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER‐lumenal molecules between cells.     

Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein.

Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum

We have identified a novel endoplasmic reticulum (ER)-resident protein, named "calumin", which is expressed in various tissues. This protein has a molecular mass of approximately 60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment, and a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of binding Ca2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and neonatal lethality, fluorometric Ca2+ imaging detected insufficient Ca2+ contents in intracellular stores and attenuated store-operated Ca2+ entry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin plays an essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision.     

Cytoplasmic targeting signals in transmembrane invariant surface glycoproteins of trypanosomes

Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family.     

Cytoplasmic phospholipase A2 antagonists inhibit multiple endocytic membrane trafficking pathways

Previous studies have suggested a role for cytosolic Ca²⁺-independent phospholipase A₂ (PLA₂) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA₂ requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA₂ antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA₂ plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules.     

Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p

Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER). We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein. Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant. The COPII components Sec23p and Sec31p and the GTP/GDP exchange factor Sec12p were required in functional form for secretion of Hsp150. Hsp150 leaves the ER in the absence of retrograde COPI traffic, and the responsible determinant is a peptide repeated 11 times in the middle of the Hsp150 sequence. Herein, we localized the sorting determinant for Sec13p-independent ER exit to the C-terminal domain. Sec13p-dependent invertase left the ER in the absence of normal Sec13p function, when fused to the C-terminal domain of Hsp150, demonstrating that this domain contained an active mediator of Sec13p-independent secretion. Thus, Hsp150 harbors two different signatures that regulate its ER exit. Our data show that transport vesicles lacking functional Sec13p can carry out ER-to-Golgi transport, but select only specific cargo protein(s) for ER exit.     

Intracellular processing of metalloprotease disintegrin ADAM12

ADAM12 has been implicated in cell-cell interactions in myogenesis and cancer, but the structure of the mature form of ADAM12 is not known, and its localization on the cell surface has been questioned. In this report, we show that full-length ADAM12 is N-glycosylated in the endoplasmic reticulum (ER) and proteolytically processed in the trans-Golgi network to an approximately 90-kDa form. The approximately 90-kDa form, which lacks the prodomain, was the predominant form present at the cell surface. Replacement of Leu(73) in the putative alpha-helical region in the prodomain with proline resulted in retention of ADAM12 in the ER and a complete lack of its processing. However, deletion of the entire pro- and metalloprotease domains did not affect the processing and trafficking of ADAM12. In contrast, replacement of the cytoplasmic domain of ADAM12 with that of ADAM9 or adding a c-Myc tag at the C terminus led to a significant increase in transport of the protein to the cell surface. These results suggest that the cytoplasmic domain of ADAM12 plays an important role in regulating ADAM12 exit from the ER. We conclude that properly folded mouse ADAM12, after passing a rate-limiting step of exit from the ER, is processed in the secretory pathway and reaches the cell surface, where it can mediate adhesion-mediated signaling.