Yip1A Structures the Mammalian Endoplasmic Reticulum

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Molecular biology of the cell (Impact Factor: 4.47). 03/2010; 21(9):1556-68. DOI: 10.1091/mbc.E09-12-1002
Source: PubMed


The structure of the endoplasmic reticulum (ER) undergoes highly regulated changes in specialized cell types. One frequently observed type of change is its reorganization into stacked and concentrically whorled membranes, but the underlying mechanisms and functional relevance for cargo export are unknown. Here, we identify Yip1A, a conserved membrane protein that cycles between the ER and early Golgi, as a key mediator of ER organization. Yip1A depletion led to restructuring of the network into multiple, micrometer-sized concentric whorls. Membrane stacking and whorl formation coincided with a marked slowing of coat protein (COP)II-mediated protein export. Furthermore, whorl formation driven by exogenous expression of an ER protein with no role in COPII function also delayed cargo export. Thus, the slowing of protein export induced by Yip1A depletion may be attributed to a proximal role for Yip1A in regulating ER network dispersal. The ER network dispersal function of Yip1A was blocked by alteration of a single conserved amino acid (E95K) in its N-terminal cytoplasmic domain. These results reveal a conserved Yip1A-mediated mechanism for ER membrane organization that may serve to regulate cargo exit from the organelle.

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Available from: Kaitlyn Dykstra, Oct 08, 2014
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    • "Moreover, Yip1A has been proposed to regulate COPI-independent retrograde transport from the GC to the ER and to be not involved in Golgi maintenance (Kano et al. 2009). More recently, it has been reported that Yip1A is involved not in Golgi architecture, but in ER structure maintenance and ER whorls formation (Dykstra et al. 2010, 2013). Our knockdown experiments did not reveal any differences in GM130 and P58 markers distribution as well as in localization of the major components of excitation–contraction machinery such as ryanodine receptor type 1, junctophilin 1, calsequestrin 1 and triadin, suggesting that Yip1B in skeletal muscle is not involved in Golgi architecture maintenance nor in targeting of triadic proteins. "
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    ABSTRACT: The mechanism of endoplasmic reticulum (ER)-Golgi complex (GC) traffic is conserved from yeast to higher animals, but the architectures and the dynamics of vesicles’ traffic between ER and GC vary across cell types and species. Skeletal muscle is a unique tissue in which ER and GC undergo a structural reorganization during differentiation that completely remodels the secretory pathway. In mature skeletal muscle, the ER is turned into sarcoplasmic reticulum, which is composed of junctional and longitudinal regions specialized, respectively, in calcium release and uptake during contraction. During skeletal muscle differentiation, GC acquires a particular fragmented organization as it appears as spots both at the nuclear poles and along the fibers. The ubiquitary-expressed Yip1A isoform has been proposed to be involved in anterograde trafficking from the ER exit sites to the cis-side of the GC and in ER and GC architecture organization. We investigated the role of Yip1 in skeletal muscle. Here we report that, following skeletal muscle development, the expression of the Yip1A decreases and is replaced by the muscle-specific Yip1B isoform. Confocal microscope analysis revealed that in adult skeletal muscle the Yip1B isoform is localized in the ER-Golgi intermediate and cis-Golgi compartments. Finally, skeletal muscle knockdown experiments in vitro and in vivo suggested that Yip1B is not involved in GC structure maintenance.
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    • "The myc-tagged Yif1A construct was cloned by PCR amplification from HeLa cDNA (Qiagen, Hilden, Germany) and inserting the PCR product into the pCS2-MT vector using EcoRI and XbaI sites. The HA-Yip1A rescue construct was created by replacing the FLAG epitope from the FLAG-Yip1A construct [10] with the HA epitope (YPYDVPDYA) using a PCR-based loop-out/loop-in modification of the QuikChange protocol (Stratagene, La Jolla, CA) and was the parent construct for all further HA-Yip1A mutations. The HA-Yip1AN/Sec61β TM was created by first using a PCR-based loop-out technique (Stratagene) to remove the TM domain region (AA 126–257) of the Yip1A construct and the TM domain from Myc- Sec61β (AA 61–97) was subcloned into the XbaI site at the C-terminus. "
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    ABSTRACT: The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.
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