scholarly journals A Functional GTPase Domain, but not its Transmembrane Domain, is Required for Function of the SRP Receptor β-subunit

1998 ◽  
Vol 142 (2) ◽  
pp. 341-354 ◽  
Author(s):  
Stephen C. Ogg ◽  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Membrane Protein ◽  
Binding Site ◽  
Protein Targeting ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Single Amino Acid ◽  
Secretory Proteins ◽  
Gtp Binding ◽  
Er Membrane

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRα and SRβ, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRβ. This notion is supported (a) by Srp102p's sequence similarity to SRβ; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRα homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.

scholarly journals Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

1992 ◽  
Vol 3 (8) ◽  
pp. 895-911 ◽  
Author(s):  
S C Ogg ◽  
M A Poritz ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Er Membrane

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.

scholarly journals SecA mediates cotranslational targeting and translocation of an inner membrane protein

2017 ◽  
Vol 216 (11) ◽  
pp. 3639-3653 ◽  
Author(s):  
Shuai Wang ◽  
Chien-I Yang ◽  
Shu-ou Shan
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Inner Membrane ◽  
Nascent Polypeptide ◽  
Inner Membrane Protein

Protein targeting to the bacterial plasma membrane was generally thought to occur via two major pathways: cotranslational targeting by signal recognition particle (SRP) and posttranslational targeting by SecA and SecB. Recently, SecA was found to also bind ribosomes near the nascent polypeptide exit tunnel, but the function of this SecA–ribosome contact remains unclear. In this study, we show that SecA cotranslationally recognizes the nascent chain of an inner membrane protein, RodZ, with high affinity and specificity. In vitro reconstitution and in vivo targeting assays show that SecA is necessary and sufficient to direct the targeting and translocation of RodZ to the bacterial plasma membrane in an obligatorily cotranslational mechanism. Sequence elements upstream and downstream of the RodZ transmembrane domain dictate nascent polypeptide selection by SecA instead of the SRP machinery. These findings identify a new route for the targeting of inner membrane proteins in bacteria and highlight the diversity of targeting pathways that enables an organism to accommodate diverse nascent proteins.

scholarly journals A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p

2003 ◽  
Vol 14 (6) ◽  
pp. 2357-2371 ◽  
Author(s):  
Sophie Chantalat ◽  
Rëgis Courbeyrette ◽  
Francesca Senic-Matuglia ◽  
Catherine L. Jackson ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
General Interest ◽  
Single Mutation ◽  
Golgi Membrane ◽  
Nucleotide Exchange

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.

scholarly journals Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

2017 ◽  
Author(s):  
Rahul Chaudhari ◽  
Vishakha Dey ◽  
Aishwarya Narayan ◽  
Shobhona Sharma ◽  
Swati Patankar
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Membrane ◽  
Membrane Protein ◽  
Host Cell ◽  
G Protein ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Acyl Carrier Protein ◽  
Secretory Proteins ◽  
Host Cell Membrane

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein dependent vesicular fusion inhibitor AlF4- and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G-protein dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl. Second, trafficking of apicoplast luminal proteins appear to be independent of G-protein coupled vesicles.

scholarly journals Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

2017 ◽  
Author(s):  
Rahul Chaudhari ◽  
Vishakha Dey ◽  
Aishwarya Narayan ◽  
Shobhona Sharma ◽  
Swati Patankar
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Membrane ◽  
Membrane Protein ◽  
Host Cell ◽  
G Protein ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Acyl Carrier Protein ◽  
Secretory Proteins ◽  
Host Cell Membrane

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein dependent vesicular fusion inhibitor AlF4- and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G-protein dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl. Second, trafficking of apicoplast luminal proteins appear to be independent of G-protein coupled vesicles.

Transposon-Disruption of a Maize Nuclear Gene, tha1, Encoding a Chloroplast SecA Homologue: In Vivo Role of cp-SecA in Thylakoid Protein Targeting

Genetics ◽  
1997 ◽  
Vol 145 (2) ◽  
pp. 467-478 ◽  
Author(s):  
Rodger Voelker ◽  
Janet Mendel-Hartvig ◽  
Alice Barkan
Keyword(s):  
Protein Targeting ◽  
Sequence Similarity ◽  
Nuclear Gene ◽  
Cytochrome F ◽  
Transposon Insertion ◽  
Nuclear Mutant ◽  
Strong Sequence Similarity

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced ≥40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

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

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

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 inSaccharomyces 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 thatLAG1 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 dgt1Δcells, 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.

scholarly journals The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor.

1994 ◽  
Vol 14 (7) ◽  
pp. 4465-4474 ◽  
Author(s):  
C Antoniewski ◽  
M Laval ◽  
A Dahan ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Site ◽  
Fat Body ◽  
Sequence Similarity ◽  
High Concentration ◽  
Direct Role ◽  
Palindromic Structure ◽  
Fbp1 Gene

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

A nematode gene required for sperm vesicle fusion

1997 ◽  
Vol 110 (9) ◽  
pp. 1073-1081 ◽  
Author(s):  
W.E. Achanzar ◽  
S. Ward
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Biological Significance ◽  
Single Amino Acid ◽  
Viral Fusion ◽  
Missense Mutations ◽  
Direct Role ◽  
C Terminus ◽  
Human Proteins

During maturation of spermatids to motile spermatozoa in Caenorhabditis elegans, large vesicles called membranous organelles (MOs) fuse with the spermatid plasma membrane. Mutations in the gene fer-1 cause abnormal spermatozoa in which the MOs do not fuse, although they abut the plasma membrane normally. Here we describe the fer-1 gene, which we found to be approximately 8.6 kb in length and to encode a 6.2 kb transcript whose expression is limited to the primary spermatocytes, the cells in which the MOs form. fer-1 is predicted to encode a 235 kDa protein which is highly charged except for a putative transmembrane domain near the C terminus. We identified the mutations associated with five fer-1 alleles, all of which are missense mutations causing single amino acid changes. FER-1 is not similar to any characterized proteins in sequence databases, nor does it contain known functional motifs other than the predicted transmembrane domain. The C-terminal transmembrane domain makes FER-1 resemble some viral fusion proteins, suggesting it may play a direct role in MO-plasma membrane fusion. FER-1 does show significant sequence similarity to several predicted human proteins of unknown function. Two of the identified fer-1 mutations are located in regions of similarity between FER-1 and two of these predicted proteins. This strengthens the biological significance of these similarities and suggests these regions of similarity represent functionally important domains of FER-1 and the human proteins.

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