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The 21-kDa GTP-binding Yptl protein (Yptlp) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Yptl antibodies block transport; this inhibition is alleviated by competition with excess purified Yptlp produced in bacteria. Fur-

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Abstract. F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gpl7) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gpl7 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gpl7 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gpl7 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gpl7 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96 % of actin-membrane binding even after extensive adsorption against cell surfaces. gpl7 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gpl7 is suggested since, in addition to the cytoplasmic localization of the actinbinding site, extracellular determinants of gpl7 are identified, gpl7 is surface-labeled by sulfo-N-hydroxysuccinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gpl7 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gpl7 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.

Abstract. Secl2p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Secl2p is inhibited in vitro by addition of microsomes isolated from a Sec12p overproducing strain . Inhibition is attributable to titration of a limiting cytosolic protein . This limitation is G NETic analysis has identified four yeast genes that are required for the formation of transport vesicles derived from the ER: SEC12 SEC13 SEC16 and SEC23 (Kaiser and Schekman, 1990). In vitro reconstitution of ER-Golgi transport vesicle formation also showed a requirement for Secl2p and Sec23p (Rexach and Schekman, 1991) . Cell fractionation and DNA sequence analysis have shown that Secl2p is an integral membrane protein, and Sec23p, Secl6p, and Secl3p are either cytosolic or peripheral membrane proteins (Nakano et al ., 1988 ;Hicke and Schekman, 1989; Kaiser, C., and N. Pryer, unpublished data) . Interactions observed among the genes encoding these proteins suggest that they could act in concert or form a multisubunit complex possibly associating with the ER membrane via Secl2p (Kaiser and Schekman, 1990) .Secl2p is a type II membrane glycoprotein with a 40 kD NH2-terminal cytosolic domain that is essential for transport, and a COON-terminal lumenal domain that is dispensable (Nakano et al., 1988 ; d'Enfert, C., C. Barlowe, S. Nishikawa, A. Nakano, and R. Schekman, manuscript submitted for publication) . The thermosensitive lethality associated with a mutation in the NH2-terminal domain of Secl2p is suppressed by overproduction of Sarlp, a 21-kD GTP-binding protein (Nakano and Muramatsu, 1989) . Sarlp is itself required for protein transport from the ER (Nakano and Muramatsu, 1989) . Cell fractionation experiments show Sarlp both in the cytosol and in tight association with a membrane fraction, however overproduction increases the fraction of Sarlp soluble in the cytosol . Membrane association of Sarlp is enhanced in vivo overcome by addition of a highly enriched fraction of soluble Sarlp, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12 . Furthermore, Sarlp binding to isolated membranes is enhanced at elevated levels of Secl2p . Sarlp-Secl2p interaction may regulate the initiation of vesicle budding from the ER.by overproduction of Secl2p (d'Enfert, C., C. Barlowe, S. Nishikawa, A. Nakano, and R. Schekman, manuscript submitted for publication) . These results suggest a structural and functional interaction of Sarlp and Secl2p.In the course of identifying cytosolic factors that stimulate protein transport from the ER in vitro, we observed that addition of a membrane fraction with elevated levels of Secl2p was inhibitory, and that a limiting cytosolic factor restored transport when provided in an enriched form . This report describes the identification of this limiting factor as Sarl...

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.

We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.

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