scholarly journals Oligomerization of a membrane protein correlates with its retention in the Golgi complex

1993 ◽  
Vol 122 (6) ◽  
pp. 1185-1196 ◽  
Author(s):  
OA Weisz ◽  
AM Swift ◽  
CE Machamer
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Cytochalasin D ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Proteolytic Digestion ◽  
Golgi Transport ◽  
Large Oligomer ◽  
Membrane Spanning ◽  
Lumenal Domain

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.

scholarly journals Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.

1993 ◽  
Vol 4 (7) ◽  
pp. 695-704 ◽  
Author(s):  
C E Machamer ◽  
M G Grim ◽  
A Esquela ◽  
S W Chung ◽  
M Rolls ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Golgi Complex ◽  
Chimeric Protein ◽  
Alpha Helix ◽  
Single Amino Acid ◽  
Golgi Retention ◽  
Polar Residues ◽  
Golgi Protein ◽  
Membrane Spanning

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps.

1995 ◽  
Vol 131 (6) ◽  
pp. 1421-1433 ◽  
Author(s):  
P A Conrad ◽  
E J Smart ◽  
Y S Ying ◽  
R G Anderson ◽  
G S Bloom
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Cholesterol Oxidase ◽  
Human Fibroblasts ◽  
Subsequent Removal ◽  
Golgi Transport ◽  
Cytoplasmic Face ◽  
Block Movement ◽  
Intermediate Compartment ◽  
In Cells

Caveolin is a protein associated with the characteristic coats that decorate the cytoplasmic face of plasma membrane caveolae. Recently it was found that exposure of human fibroblasts to cholesterol oxidase (CO) rapidly induces caveolin to redistribute to the ER and then to the Golgi complex, and that subsequent removal of CO allows caveolin to return to the plasma membrane (Smart, E. J., Y.-S. Ying, P. A. Conrad, R. G. W. Anderson, J. Cell Biol. 1994, 127:1185-1197). We now present evidence that caveolin normally undergoes microtubule-dependent cycling between the plasma membrane and the Golgi. In cells that were treated briefly with nocodazole and then with a mixture of nocodazole plus CO, caveolin relocated from the plasma membrane to the ER and then to the ER/Golgi intermediate compartment (ERGIC), but subsequent movement to the Golgi was not observed. Even in the absence of CO, nocodazole caused caveolin to accumulate in the ERGIC. Nocodazole did not retard the movement of caveolin from the Golgi to the plasma membrane after removal of CO. Incubation of cells at 15 degrees followed by elevation of the temperature to 37 degrees caused caveolin to accumulate first in the ERGIC and then in the Golgi, before finally reestablishing its normal steady state distribution predominantly in plasma membrane caveolae. In cells released from a 15 degrees block, movement of caveolin from the Golgi to the plasma membrane was not inhibited by nocodazole. Taken together, these results imply that caveolin cycles constitutively between the plasma membrane and the Golgi by a multi-step process, one of which, ERGIC-to-Golgi transport, requires microtubules. This novel, bidirectional pathway may indicate roles for microtubules in the maintenance of caveolae, and for caveolin in shuttling fatty acids and cholesterol between the plasma membrane and the ER/Golgi system.

scholarly journals A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein.

1991 ◽  
Vol 115 (1) ◽  
pp. 19-30 ◽  
Author(s):  
A M Swift ◽  
C E Machamer
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Alpha Helix ◽  
Single Amino Acid ◽  
Transfected Cells ◽  
E1 Protein ◽  
Golgi Region ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Retention Signal

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.

scholarly journals Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells

1998 ◽  
Vol 143 (6) ◽  
pp. 1485-1503 ◽  
Author(s):  
Koret Hirschberg ◽  
Chad M. Miller ◽  
Jan Ellenberg ◽  
John F. Presley ◽  
Eric D. Siggia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Rate Constants ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Single Cells ◽  
Cell Periphery ◽  
Protein Traffic ◽  
Golgi Transport

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.

scholarly journals Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

2005 ◽  
Vol 169 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Daniela A. Sahlender ◽  
Rhys C. Roberts ◽  
Susan D. Arden ◽  
Giulietta Spudich ◽  
Marcus J. Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rna Interference ◽  
Vesicular Stomatitis Virus ◽  
Protein Interaction ◽  
Golgi Complex ◽  
Myosin Vi ◽  
Binding Partner ◽  
Binding Partners ◽  
Vesicular Stomatitis ◽  
Golgi Organization

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suman Pokhrel ◽  
Benjamin R. Kraemer ◽  
Scott Burkholz ◽  
Daria Mochly-Rosen
Keyword(s):  
Amino Acid ◽  
Severe Disease ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Severe Respiratory Distress ◽  
S Protein ◽  
Individual Sequences ◽  
Natural Variants ◽  
Novel Coronavirus ◽  
The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

Lateral Mobility of Integrin αIIbβ3 (Glycoprotein IIb/IIIa) in the Plasma Membrane of a Human Megakaryocyte

1997 ◽  
Vol 77 (01) ◽  
pp. 143-149 ◽  
Author(s):  
Annelies Schootemeijer ◽  
Gijsbert van Willigen ◽  
Hans van der Vuurst ◽  
Leon G J Tertoolen ◽  
Siegfried W De Laat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Recovery After Photobleaching ◽  
Cell Activation ◽  
Lateral Diffusion ◽  
Cytochalasin D ◽  
Lag Phase ◽  
Integrin Αiibβ3 ◽  
Cell Matrix ◽  
Cytoplasmic Actin ◽  
Mobile Fraction

SummaryThe migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin αIIbβ3 (glycoprotein Ilb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the P3 subunit. The diffusion of P3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 X10'9 cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 μM) or α-thrombin (10 U/ml) at 22° C induced transient decreases in both parameters reducing D to 0.21 X 10‘9 cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, whereas cytochalasin D completely abolished the decrease in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile αIIbβ3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.

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