Location of light-repressible, small GTP-binding protein of the YPT/rab family in the growing zone of etiolated pea stems.

YPT/rab proteins are ras-like small GTP-binding proteins that serve as key regulators of vesicular transport. The mRNA levels of two YPT/rab genes in pea plants are repressed by light, with the process mediated by phytochrome. Here, we examined the mRNA expression and the location of the two proteins, pra2- and pra3-encoded proteins, using monoclonal antibodies. The pra2 and pra3 mRNA levels were highest in the stems of dark-grown seedlings. The corresponding proteins were found in the cytosol and the membranes of the stems. Most of the pra2 protein was in the growing internodes, especially in the growing region, but the pra3 protein was widespread. These results suggest that the pra2 protein is important for vesicular transport in stems, possibly contributing to stem growth in the dark, and that the pra3 protein is important for general vesicular transport. The amounts of pra2 and pra3 proteins decreased with illumination. The decrease in these proteins may be related to the phytochrome-dependent inhibition of stem growth that occurs in etiolated pea seedlings.

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J. B. & Rothman, J. E. (1992) Nature (London) 360, 352–354; Donaldson, J. G., Finazzi, D. & Klausner, R. D. (1992) Nature (London) 360, 350–352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305–309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE–Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (≈670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of ≈190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

Formation and Turnover of NSF- and SNAP-containing “Fusion” Complexes Occur on Undocked, Clathrin-coated Vesicle–derived Membranes

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle–derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

Differential Roles of Syntaxin 7 and Syntaxin 8 in Endosomal Trafficking

To understand molecular mechanisms that regulate the intricate and dynamic organization of the endosomal compartment, it is important to establish the morphology, molecular composition, and functions of the different organelles involved in endosomal trafficking. Syntaxins and vesicle-associated membrane protein (VAMP) families, also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), have been implicated in mediating membrane fusion and may play a role in determining the specificity of vesicular trafficking. Although several SNAREs, including VAMP3/cellubrevin, VAMP8/endobrevin, syntaxin 13, and syntaxin 7, have been localized to the endosomal membranes, their precise localization, biochemical interactions, and function remain unclear. Furthermore, little is known about SNAREs involved in lysosomal trafficking. So far, only one SNARE, VAMP7, has been localized to late endosomes (LEs), where it is proposed to mediate trafficking of epidermal growth factor receptor to LEs and lysosomes. Here we characterize the localization and function of two additional endosomal syntaxins, syntaxins 7 and 8, and propose that they mediate distinct steps of endosomal protein trafficking. Both syntaxins are found in SNARE complexes that are dissociated by α-soluble NSF attachment protein and NSF. Syntaxin 7 is mainly localized to vacuolar early endosomes (EEs) and may be involved in protein trafficking from the plasma membrane to the EE as well as in homotypic fusion of endocytic organelles. In contrast, syntaxin 8 is likely to function in clathrin-independent vesicular transport and membrane fusion events necessary for protein transport from EEs to LEs.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

Transcytosis of α1-acidic glycoprotein in the continuous microvascular endothelium

By using perfusions and bolus administration, coupled with postembedding immunocytochemical procedures, we have identified the structures involved in the transport of derivatized orosomucoid (α1-acidic glycoprotein) across the continuous microvascular endothelium of the murine myocardium. Our findings indicate that: (i) monomeric orosomucoid binds to the luminal surface of the endothelium; (ii) it is restricted to caveolae during its transport across the endothelium; (iii) it is detected in the perivascular spaces at early time points (by 1 min) and in larger quantities at later time points (>5 min) from the beginning of its perfusion or its intravascular administration; (iv) no orosomucoid molecules are found in the intercellular junctions or at the abluminal exits of interendothelial spaces; and (v) the vesicular transport of orosomucoid is strongly inhibited by N-ethylmaleimide (>80%). Because, by size and shape, the orosomucoid qualifies as a preferential probe for the postulated small pore system, our results are discussed in relation to the pore theory of capillary permeability.

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.

Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility.

A framework for interpreting the leucine-rich repeats of the Listeria internalins

The surface protein InlB of the bacterial pathogen Listeria monocytogenes is required for inducing phagocytosis in various nonphagocytic mammalian cell types in vitro. InlB causes tyrosine phosphorylation of host cell adaptor proteins, activation of phosphoinositide 3-kinase, and rearrangements of the actin cytoskeleton. These events lead to phagocytic uptake of the bacterium by the host cell. InlB belongs to the internalin family of Listeria proteins, which also includes InlA, another surface protein involved in host cell invasion. The internalins are the largest class of bacterial proteins containing leucine-rich repeats (LRR), a motif associated with protein–protein interactions. The LRR motif is found in a functionally diverse array of proteins, including those involved in the plant immune system and in the mammalian innate immune response. Structural and functional interpretations of the sequences of internalin family members are presented in light of the recently determined x-ray crystal structure of the InlB LRR domain.

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δ cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.