“…Of 133 degraded proteins identified in the ‘high’ or ‘medium confidence’ shortlist, 11 also had a role in protein transport (). These included ADP-Ribosylation Factor Guanine Nucleotide-Exchange Factor 2 (ARFGEF2), important for trans-Golgi network integrity and trafficking [46–48]; GRIP1 Associated Protein 1 (GRIPAP1), involved in the regulation of endosomal recycling [49]; Secretion Associated Ras Related GTPase 1 paralogues (SAR1A and SAR1B), components of coat protein complex II (COPII) that facilitate transport of proteins from the ER [50, 51]; and Lectin mannose binding 2-like (LMAN2L), with hypothesized roles in the early secretory pathway [52, 53]. ARFGEF2 and GRIPAP1 have been implicated in protein expression at the plasma membrane.…”
The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host’s endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.
“…Of 133 degraded proteins identified in the ‘high’ or ‘medium confidence’ shortlist, 11 also had a role in protein transport (). These included ADP-Ribosylation Factor Guanine Nucleotide-Exchange Factor 2 (ARFGEF2), important for trans-Golgi network integrity and trafficking [46–48]; GRIP1 Associated Protein 1 (GRIPAP1), involved in the regulation of endosomal recycling [49]; Secretion Associated Ras Related GTPase 1 paralogues (SAR1A and SAR1B), components of coat protein complex II (COPII) that facilitate transport of proteins from the ER [50, 51]; and Lectin mannose binding 2-like (LMAN2L), with hypothesized roles in the early secretory pathway [52, 53]. ARFGEF2 and GRIPAP1 have been implicated in protein expression at the plasma membrane.…”
The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host’s endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.
“…1b, c ). We additionally took advantage of RPE1 cells natively expressing HaloTag-Sec23a, which we described previously 67 . Fig.…”
Section: Resultsmentioning
confidence: 99%
“…In light of these opposing findings, we developed an alternative approach to examine ER export under differing nutrient conditions that avoids the use of artificial cargoes and instead focuses on endogenous ERGIC-53, a well-characterized COPII cargo that guides several glycoproteins during their anterograde transport 37 , 38 . For these studies, we used CRISPR/Cas9-edited cells expressing SNAP-tag-ERGIC-53 from its native locus 67 and following dye-labeling, conducted highly inclined thin illumination (HILO) imaging to monitor changes in its fluorescence intensity at newly formed peripheral sites. We found that SNAP-tag-ERGIC-53 accumulated approximately 60% more slowly following acute nutrient deprivation, as compared to either nutrient replete conditions or when nutrients were absent for a prolonged period (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…CRISPR/Cas9-mediated genome editing of human hTERT-immortalized RPE1 cells (CRL-4000 from ATCC) grown in DMEM:F-12 media (nutrient replete) was conducted as described previously 67 , 74 . Briefly, cells were transfected with purified Cas9, a specific gRNA, and a homology directed repair (HDR) template.…”
Section: Methodsmentioning
confidence: 99%
“…The following guide RNA (gRNA) sequences were used: 5’-GACGGGACCGTCTGGGGCGG-3’ (targeting Sec16a), 5’-CTTTAACTTCATCCTGCTAA-3’ (targeting Sec31a), 5’- ATCCAACTGTCCGTTCATGG-3’ (targeting TFG), and 5’- TCTACCACAGAATAACACCC-3’ (targeting GRASP65). Other edited cell lines have been described elsewhere 67 . Clonal populations were isolated using fluorescence activated cell sorting (FACS), which was followed by live cell imaging to confirm the proper distribution of each fusion protein.…”
Co-assembly of the multilayered coat protein complex II (COPII) with the Sar1 GTPase at subdomains of the endoplasmic reticulum (ER) enables secretory cargoes to be concentrated efficiently within nascent transport intermediates, which subsequently deliver their contents to ER-Golgi intermediate compartments. Here, we define the spatiotemporal accumulation of native COPII subunits and secretory cargoes at ER subdomains under differing nutrient availability conditions using a combination of CRISPR/Cas9-mediated genome editing and live cell imaging. Our findings demonstrate that the rate of inner COPII coat recruitment serves as a determinant for the pace of cargo export, irrespective of COPII subunit expression levels. Moreover, increasing inner COPII coat recruitment kinetics is sufficient to rescue cargo trafficking deficits caused by acute nutrient limitation. Our findings are consistent with a model in which the rate of inner COPII coat addition acts as an important control point to regulate cargo export from the ER.
Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane‐bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII‐mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site‐directed mutagenesis to generate a dominant‐negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER‐Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER‐to‐Golgi protein secretory pathway in green algae.
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