Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway

Cell ◽  
1990 ◽  
Vol 61 (4) ◽  
pp. 723-733 ◽  
Author(s):  
Chris A. Kaiser ◽  
Randy Schekman
Keyword(s):  
Secretory Pathway ◽  
Vesicle Formation ◽  
Transport Vesicle

scholarly journals SED4 encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation.

1995 ◽  
Vol 131 (2) ◽  
pp. 325-338 ◽  
Author(s):  
R E Gimeno ◽  
P Espenshade ◽  
C A Kaiser
Keyword(s):  
Secretory Pathway ◽  
Silent Mutation ◽  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Golgi Transport ◽  
Transport Vesicles ◽  
Endoplasmic Reticulum Protein ◽  
Transport Defect ◽  
Transport Vesicle ◽  
Er Membrane

SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage-dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.

scholarly journals Specific Sterols Required for the Internalization Step of Endocytosis in Yeast

1999 ◽  
Vol 10 (11) ◽  
pp. 3943-3957 ◽  
Author(s):  
Alan L. Munn ◽  
Antje Heese-Peck ◽  
Brian J. Stevenson ◽  
Harald Pichler ◽  
Howard Riezman
Keyword(s):  
Biosynthetic Pathway ◽  
Secretory Pathway ◽  
Fluid Phase ◽  
Sterol Composition ◽  
Yeast Cells ◽  
Vesicle Formation ◽  
Wild Type ◽  
Major Sterol ◽  
Wild Type Yeast ◽  
Late Part

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Δ, erg6Δ, anderg2Δerg6Δ) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergΔ mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergΔ mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37°C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

scholarly journals The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.

1992 ◽  
Vol 118 (6) ◽  
pp. 1321-1332 ◽  
Author(s):  
B W Wattenberg ◽  
T J Raub ◽  
R R Hiebsch ◽  
P J Weidman
Keyword(s):  
Protein Transport ◽  
Vesicle Formation ◽  
Attachment Protein ◽  
Direct Role ◽  
Transport Vesicles ◽  
Sensitive Factor ◽  
Fusion Site ◽  
Transport Vesicle ◽  
Target Membrane ◽  
A Cell

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.

scholarly journals An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

2021 ◽  
Vol 118 (35) ◽  
pp. e2101287118
Author(s):  
Yan Huang ◽  
Haidi Yin ◽  
Baiying Li ◽  
Qian Wu ◽  
Yang Liu ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Vesicular Trafficking ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Cargo Proteins ◽  
Er Exit Sites

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

Transport vesicle formation in plant cells

2009 ◽  
Vol 12 (6) ◽  
pp. 660-669 ◽  
Author(s):  
Inhwan Hwang ◽  
David G Robinson
Keyword(s):  
Plant Cells ◽  
Vesicle Formation ◽  
Transport Vesicle

scholarly journals Poliovirus Infection Transiently Increases COPII Vesicle Budding

2012 ◽  
Vol 86 (18) ◽  
pp. 9675-9682 ◽  
Author(s):  
Meg Trahey ◽  
Hyung Suk Oh ◽  
Craig E. Cameron ◽  
Jesse C. Hay
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Vesicle Formation ◽  
Vesicle Budding ◽  
Precursor Pool ◽  
Copii Vesicle ◽  
Early Increase ◽  
Vesicle Biogenesis ◽  
Intermediate Compartment ◽  
Copii Vesicles

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication “organelles” of PV (N. Y. Hsu et al., Cell 141:799–811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.

scholarly journals Cvt9/Gsa9 Functions in Sequestering Selective Cytosolic Cargo Destined for the Vacuole

2001 ◽  
Vol 153 (2) ◽  
pp. 381-396 ◽  
Author(s):  
John Kim ◽  
Yoshiaki Kamada ◽  
Per E. Stromhaug ◽  
Ju Guan ◽  
Ann Hefner-Gravink ◽  
...  
Keyword(s):  
Temperature Sensitive ◽  
Vesicle Formation ◽  
Sensitive Mutant ◽  
Direct Role ◽  
Vacuole Membrane ◽  
Cytoplasmic Material ◽  
Transport Vesicle ◽  
Membrane Compartment ◽  
Selective Delivery

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection. We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

scholarly journals Biogenesis of γ-secretase early in the secretory pathway

2007 ◽  
Vol 179 (5) ◽  
pp. 951-963 ◽  
Author(s):  
Jinoh Kim ◽  
Bertrand Kleizen ◽  
Regina Choy ◽  
Gopal Thinakaran ◽  
Sangram S. Sisodia ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Evidence ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Transport Vesicles ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
Transient Intermediate ◽  
Important Enzyme

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.

scholarly journals δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

2016 ◽  
Vol 113 (25) ◽  
pp. 6916-6921 ◽  
Author(s):  
Eric C. Arakel ◽  
Kora P. Richter ◽  
Anne Clancy ◽  
Blanche Schwappach
Keyword(s):  
Secretory Pathway ◽  
Conformational Dynamics ◽  
Functional Complementation ◽  
Vesicle Formation ◽  
Membrane Recruitment ◽  
Early Secretory Pathway ◽  
Packing Defects ◽  
Clathrin Adaptor ◽  
Dynamics And Function ◽  
And Function

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

2007 ◽  
Vol 409 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Satoshi Takida ◽  
Yusuke Maeda ◽  
Taroh Kinoshita
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Direct Evidence ◽  
Temperature Sensitive ◽  
Early Secretory Pathway ◽  
Cargo Proteins ◽  
Transport Vesicle ◽  
A Cell ◽  
P24 Proteins

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

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