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M G Roth

Publications and source records attributed to M G Roth.

At least 19 recordsLinked to original sources
PubMed2001

Internalization-competent influenza hemagglutinin mutants form complexes with clathrin-deficient multivalent AP-2 oligomers in live cells.

Most membrane proteins are endocytosed through clathrin-coated pits via AP-2 adaptor complexes. However, little is known about the interaction of internalization signals with AP-2 in live cells in the absence of clathrin lattices. To investigate this issue, we employed cells cotransfected with pairs of antigenically distinct influenza hemagglutinin (HA) mutants containing different internalization signals of the YXXZ family. To enable studies on the possible association of the naturally trimeric HAs into higher order complexes via binding to AP-2, we exploited the inability of HAs from different influenza strains to form mutual trimers. Thus, we coexpressed HA pairs from different strains (Japan and X:31) bearing similar cytoplasmic tails mutated to include internalization signals. Using antibody-mediated immunofluorescence co-patching on live cells, we demonstrate that internalization-competent HA mutants form higher order complexes and that this clustering depends on the strength of the internalization signal. The clustering persisted in cells treated with hypertonic medium to disperse the clathrin lattices, as validated by co-immunoprecipitation experiments. The clustering of HAs bearing strong internalization signals appears to be mediated via binding to AP-2, as indicated by (i) the coprecipitation of alpha-adaptin with these HAs, even in hypertonically treated cells; (ii) the co-localization (after hypertonic treatment) of AP-2 with antibody-mediated patches of these mutants; and (iii) the dispersal of the higher order HA complexes following chlorpromazine treatment, which removes AP-2 from the plasma membrane. These results suggest that even in the absence of clathrin lattices, AP-2 exists in multivalent complexes capable of simultaneously binding several internalization signals from the same family.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin.

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.

Animals↗
PubMed2000

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

3T3 Cells↗
PubMed2000

A screen of random sequences for those that alter the trafficking of the influenza virus hemagglutinin in vivo.

In order to determine if the sequence patterns known to specify internalization represent the majority of possible internalization signals, we identified random sequences capable of causing a reporter protein to be internalized at least several-fold faster than the rate of non-selective internalization of membrane by clathrin-coated pits. A library of influenza hemagglutinin (HA) proteins, bearing short random sequences in place of the wild-type cytoplasmic domain, was prepared in recombinant SV40 virus. The library was expressed and screened for HAs that could internalize anti-HA antibody from the medium. The cytoplasmic sequences of the selected proteins were determined. From a small sample of sequences we detected several that did not resemble those previously identified. The known internalization signals must represent only a subset of the sequences that can serve as internalization signals.

Amino Acid Motifs↗
PubMed2000

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion.

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

Animals↗
PubMed1999

An internalization-competent influenza hemagglutinin mutant causes the redistribution of AP-2 to existing coated pits and is colocalized with AP-2 in clathrin free clusters.

Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that approximately 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin and AP-2 at the plasma membrane of intact cells, while wild-type HA (which is excluded from coated pits) does not colocalize with either protein. Clathrin and AP-2 clusters were saturated when HA+8 was overexpressed, and this was accompanied by a redistribution of AP-2 into existing coated pits. However, de novo coated pit formation was not observed. In nontreated cells, the number of clusters of clathrin or AP-2 colocalized with HA+8 was always comparable. Hypertonic treatment which disperses the clathrin lattices resulted in more clusters containing AP-2 and HA+8 than clathrin and HA+8. Less colocalization of HA+8 with clathrin was also observed after cytosol acidification, which causes the formation of deeply invaginated pits, where the HA+8 may be inaccessible to extracellular labeling by antibodies, and blocks coated vesicle budding. However, cytosol acidification elevated the number of clusters containing both HA+8 and AP-2, suggesting an increase in their level of association outside of the deep invaginations. Our results imply that AP-2 and HA+8 can colocalize in clusters devoid of clathrin, at least in cells treated to alter the clathrin lattice structure. Although we cannot ascertain whether this also occurs in untreated cells, we propose that AP-2 binding to membrane proteins carrying internalization signals can occur prior to the binding of AP-2 to clathrin. While such complexes can in principle serve to recruit clathrin for the formation of new coated pits, the higher affinity of the internalization signals for clathrin-associated AP-2 [Rapoport, I., et al. (1997) EMBO J. 16, 2240-2250] makes it more likely that once the AP-2-membrane protein complexes form, they are quickly recruited into existing coated pits.

Animals↗
PubMed1999

Hierarchy of sorting signals in chimeras of intestinal lactase-phlorizin hydrolase and the influenza virus hemagglutinin.

Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.

Amino Acid Sequence↗
PubMed1999

Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated.

Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Madin-Darby canine kidney cell DRMs (other than cytoskeletal contaminants) could be labeled with [3H]palmitate. Association of influenza hemagglutinin with DRMs required all three of its palmitoylated Cys residues. Prenylated proteins, detected by [3H]mevalonate labeling or by blotting for Rap1, Rab5, Gbeta, or Ras, were excluded from DRMs. Rab5 and H-Ras each contain more than one lipid group, showing that hydrophobicity alone does not target multiply lipid-modified proteins to DRMs. Partitioning of covalently linked saturated acyl chains into liquid-ordered phase domains is likely to be an important mechanism for targeting proteins to DRMs.

Acylation↗
PubMed1999

Phospholipase D as an effector for ADP-ribosylation factor in the regulation of vesicular traffic.

A mammalian phospholipase D (PLD) activity that is stimulated by ADP-ribosylation factor (ARF) has been identified in Golgi-enriched membrane fractions. This activity is due to the PLD1 isoform and evidence from several laboratories indicates that PLD1 is important for the polymerization of vesicle coat proteins on membranes. When expressed in Chinese hamster ovary cells, PLD1 localized to dispersed small vesicles that overlapped with the location of the ERGIC53 protein, a marker for the endoplasmic reticulum (ER)-Golgi intermediate compartment. Cells having increased PLD1 expression had accelerated anterograde and retrograde transport between the ER and Golgi. Membranes from cells having elevated PLD1 activity bound more COPI, ARF, and ARF-GTPase activating protein. These membranes also produced more COPI vesicles than did membranes from control cells. It is likely that PLD1 participates in both positive and negative feedback regulation of the formation of COPI vesicles and is important for controlling the rate of this process.

ADP-Ribosylation Factors↗
PubMed1999

Lipid regulators of membrane traffic through the Golgi complex.

Enzymes that modify phospholipids play necessary, but poorly understood, roles in constitutive membrane traffic. Local production of specific phosphoinositides is required for endocytosis and regulated exocytosis, and enzymes that produce and consume phosphoinositides are components of post-Golgi membrane vesicles. Both biochemical and genetic data indicate that regulation of the membrane content of phosphatidic acid, diacylglycerol and phosphoinositides is necessary for protein traffic from the Golgi complex. Evidence for a regulatory role for lipids earlier in the constitutive secretory pathway is more limited and controversial. Although the mechanisms that regulate traffic between the endoplasmic reticulum and Golgi might be qualitatively different from those that control later membrane transport pathways, recent studies suggest that production of specific lipids is important for transport both into and out of the Golgi. As discussed in this article, one potential mechanism for the involvement of lipids is to control the GTPase cycle of a small GTP-binding protein, ARF (ADP-ribosylation factor).

Biological Transport↗
PubMed1999

Amino acid sequence requirements of the transmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion.

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

Amino Acid Sequence↗
PubMed1998

Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells.

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.

Amino Acid Sequence↗
PubMed1998

Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells.

The composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. In MDCK cells, the influenza virus hemagglutinin is sorted in the trans-Golgi network into detergent-insoluble, glycosphingolipid-enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. Site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. The region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. Several mutants were found to enter detergent-insoluble membranes but were not properly sorted. Replacement of transmembrane residues 520 and 521 with alanines converted the 2A520 mutant hemagglutinin into a basolateral protein. Depleting cell cholesterol reduced the ability of wild-type hemagglutinin to partition into detergent-insoluble membranes but had no effect on apical or basolateral sorting. In contrast, cholesterol depletion allowed random transport of the 2A520 mutant. The mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent-insoluble membranes were present. Apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent-insoluble membrane domains. Entry into these domains appears necessary, but not sufficient, for apical sorting.

Amino Acid Sequence↗
PubMed1998

Susceptibility of epithelial cells to Pseudomonas aeruginosa invasion and cytotoxicity is upregulated by hepatocyte growth factor.

Normal cell polarity protects epithelial cells against Pseudomonas aeruginosa invasion and cytotoxicity. Using epithelial cell clones with selective defects in sorting of membrane constituents, and using hepatocyte growth factor pretreatment, we found that polarized susceptibility to P. aeruginosa can be altered without disrupting tight junctions. The results also showed that cellular susceptibility factors for invasion and cytotoxicity are not the same, although both are localized to the basolateral cell surface in polarized epithelial cells.

Animals↗