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H P Moore

Publications and source records attributed to H P Moore.

At least 37 records · Page 2Linked to original sources
PubMed1994

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II.

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.

Adenosine Triphosphate↗
PubMed1994

Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.

We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, ISC, in microA/cm2) and apical (ap) Cl permeability (PCl) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. PCl and PK were measured by permeabilizing the bl or ap membrane with nystatin. PCl was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 35SO4-labeled glycosaminoglycan [GAG], a fluid-phase marker of Golgi-derived vesicles) was used to measure exocytosis at both ap and bl membranes. At rest, ISC = 3.6, PK = 0.8 x 10(-6), PCl = 2.1 x 10(-6) with SPQ and 2.4 x 10(-6) electrically, and there was constitutive GAG secretion (i.e., exocytosis) to both ap and bl sides (bl > 2 x ap). Carbachol (C) increased: ISC (delta = 18.6), PK (6.5x), PCl (1.8-2.9x), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (delta = 29), PCl (5.5-11x) and ap exocytosis (1.5-2x), but had no effect on PK or bl exocytosis. Synergistic effects on ISC occurred when C was added to F-treated cells but not vice versa, even though the characteristic effects of F+C on PCl, PK, and/or GAG secretion were identical to those exhibited when stimulants were added individually. Cl secretion results from coordinated activation of channels at ap and bl membranes, and exocytosis may play a role in these events.

Carbachol↗
PubMed1993

Molecular analysis of SAR1-related cDNAs from a mouse pituitary cell line.

Vesicular transport between the endoplasmic reticulum (ER) and the Golgi in the yeast Saccharomyces cerevisiae requires a Ras-like, small GTP-binding protein, Sar1p [1-3]. Whether a functional homologue operates in export from the ER in mammalian cells is unknown, nor is it clear if transport in other branches of the secretory pathway requires member(s) of a gene family. In this study, we used a PCR approach to examine the complexity of SAR1-related sequences expressed in mammalian cells that possess multiple secretory pathways. Amplification of cDNA sequences from rodent pituitary cells with primers corresponding to two conserved GTP binding domains of Sar1p yielded several clones with sequences homologous to Sar1 and/or the closely related ADP-ribosylation factor (ARF) family. Of these, only two showed closer homologies to S. cerevisiae Sar1 than members of the ARF family and are designated as mSARa and mSARb. Northern blot analysis shows that mSARa is expressed in most tissues including liver, heart, brain, skeletal muscle and kidney. In contrast, mSARb is preferentially expressed in skeletal muscle and liver. The full-length cDNA of mSARa isolated from a mouse pituitary AtT-20 cDNA library encodes a protein of 198 amino acids, and is 61.6% identical to Sar1p from S. cerevisiae. Thus in contrast to the large rab family of GTP-binding proteins, vesicular transport in mammalian cells appears to be mediated by a relatively small number of Sar1-related proteins.

Amino Acid Sequence↗
PubMed1993

Gene cloning and characterization of a GTP-binding Rab protein from mouse pituitary AtT-20 cells.

The Rab proteins constitute a family of Ras-like, low-molecular-weight GTP-binding proteins that are involved in vesicular transport in mammalian cells. Several members of this family have been localized to specific cellular compartments, indicating that they may control vesicular transfer between discrete portions of the endomembrane systems. To examine the complexity of the Rab proteins produced in cells with both regulated and constitutive secretory pathways, we used a PCR-based strategy to clone members of this gene family from rodent pituitary. Here, we report the identification and studies of one of the clones, RAB18. The full-length cDNA clone from a mouse pituitary AtT-20 cDNA library encodes a 23.5-kDa protein with four consensus GTP-binding domains. The deduced amino acid sequence is 42% and 40% identical to Rab2 and Sec4p, respectively, and contains sequence motifs characteristic of the Rab family. Northern blot analysis shows that the level of expression is high in the brain, moderate in the pituitary, and low in the liver. Rab18 may be involved in membrane traffic events during biogenesis and/or recycling steps of secretory vesicle components.

Amino Acid Sequence↗
PubMed1993

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Small GTP-binding proteins of the YPT/SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking. In mammals, Rab8 and Rab10 are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae. To study the localization of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus. The feasibility of this method was tested by using yeast SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-type SEC4, indicating that the modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection. In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the ruffling areas. In contrast, epitope-tagged Rab10 expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region. By light microscopy, the staining partially overlapped with that of a Golgi marker, beta-COP. Thus, despite the high degree homology of Rab8 and Rab10 (66% identity), the two proteins are localized to distinct cellular compartments. This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family.

Animals↗
PubMed1992

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

The establishment of polarized membrane traffic in Xenopus laevis embryos.

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.

Animals↗
PubMed1991

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.

Animals↗
PubMed1991

Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).

Animals↗
PubMed1991

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

Amino Acid Sequence↗
PubMed1990

The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes.

Protein secretion is blocked in Xenopus oocytes arrested at second meiotic metaphase. In this report, we show that secretion becomes blocked coincident with germinal vesicle breakdown (GVBD). Transport through the metaphase-arrested oocyte's secretory pathway continues unimpeded until proteins reach the trans-Golgi. These conclusions are drawn from experiments using exogenous prolactin and vesicular stomatitis virus G protein (VSV G) encoded by SP6 transcripts and endogenous glycosaminoglycan (GAG) chains initiated on beta-D-4-methylumbelliferyl-xyloside. From the initiation of maturation with progesterone until GVBD, secretion of prolactin synthesized before the start of maturation is comparable to secretion in immature oocytes, but after GVBD secretion of prolactin declines approximately 63% in the first hour. Not all steps in the secretory pathway are blocked when oocytes mature. Since VSV G protein acquires resistance to endo H digestion with equal efficiency in immature oocytes (arrested in first meiotic prophase) and matured oocytes (arrested in second meiotic metaphase), we conclude that transport of this protein from the ER to the Golgi is not inhibited at meiotic metaphase. Using [35S]sulfate to label xyloside-initiated GAG chains we find that transport of GAG chains from the trans-Golgi to the cell surface is 15-fold lower in matured oocytes than in immature oocytes. Examination of the size of GAG chains by SDS-PAGE and HPLC indicates that matured oocytes produce GAG chains significantly larger than GAG chains from immature oocytes. This increase in size suggests that GAG chains from matured oocytes have a longer residence time in the trans-Golgi than GAG chains from immature oocytes. Hence, part of the block to secretion in metaphase-arrested oocytes could be an inhibition of vesicle budding from the trans-Golgi.

Animals↗
PubMed1990

Membrane traffic between secretory compartments is differentially affected during mitosis.

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Vesicle formation in the Golgi apparatus.

In this paper we examine the mechanics of vesicle budding from the Golgi apparatus. We propose a model for this process based on the notion that molecular surfactants can release the elastic energy stored in the lipid bilayer. The same physical process may drive other vesiculation processes, including coated vesicle formation and budding of enveloped viruses from the plasma membrane.

Golgi Apparatus↗
PubMed1989

Molecular sorting in the secretory pathway.

Proteins can be secreted from animal cells by either a constitutive or a regulated pathway; those destined for regulated secretion are actively sorted into dense-core secretory granules. Although sorting is generally assumed to be accomplished by specific carriers, the nature of these carriers remains elusive. In this study, peptide hormones were used as affinity ligands to purify a set of 25-kilodalton proteins from canine pancreatic tissue. Their ligand specificities and patterns of expression have the characteristics of sorting carriers.

Animals↗
PubMed1989

Spatial segregation of the regulated and constitutive secretory pathways.

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.

Adrenocorticotropic Hormone↗