PGRMC1 acts as a size-selective cargo receptor to drive ER-phagic clearance of mutant prohormones

The reticulon-3 (RTN3)-driven targeting complex promotes clearance of misfolded prohormones from the endoplasmic reticulum (ER) for lysosomal destruction by ER-phagy. Because RTN3 resides in the cytosolic leaflet of the ER bilayer, the mechanism of selecting misfolded prohormones as ER-phagy cargo on the luminal side of the ER membrane remains unknown. Here we identify the ER transmembrane protein PGRMC1 as an RTN3-binding partner. Via its luminal domain, PGRMC1 captures misfolded prohormones, targeting them for RTN3-dependent ER-phagy. PGRMC1 selects cargos that are smaller than the large size of other reported ER-phagy substrates. Cargos for PGRMC1 include mutant proinsulins that block secretion of wildtype proinsulin through dominant-negative interactions within the ER, causing insulin-deficiency. Chemical perturbation of PGRMC1 partially restores WT insulin storage by preventing ER-phagic degradation of WT and mutant proinsulin. Thus, PGRMC1 acts as a size-selective cargo receptor during RTN3-dependent ER-phagy, and is a potential therapeutic target for diabetes.

Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

Centrosomes consist of two centrioles surrounded by pericentriolar material and are the main microtubule organizing centre in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, a master regulator of centrosome biogenesis. In an evolutionarily conserved process, altered centrosome numbers cause a p53-dependent growth arrest through mechanisms that are still poorly defined. To gain insights into this process, we used a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest after chemically altering PLK4 activity to cause too many or too few centrosomes. We identify TRIM37 as a key mediator of growth arrest when PLK4 activity is partially or fully inhibited but is not required for growth arrest triggered by supernumerary centrosomes. Moreover, this activity is independent of its role as an E3 ligase and distinct from other TRIM37 functions described to date. We propose that altered PLK4 activity itself can signal growth arrest.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. While centrosomes facilitate bipolar spindle formation, the individual functions of the centriole and PCM in mitosis remain elusive. Herein, we show the redundant roles of the centriole and PCM in bipolar spindle formation in human cells. Upon depletion of the PCM scaffold components, pericentrin and CDK5RAP2, centrioles remained able to recruit CEP192 onto their walls, which was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the chemical perturbation of polo-like kinase 1, a critical kinase for PCM assembly, efficiently suppressed the proliferation of various cancer cell lines from which centrioles were removed. Overall, these data suggest that the centriole and PCM cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation in human cells.

Robustness in glycosylation systems: effect of modified monosaccharides, acceptor decoys and azido sugars on cellular nucleotide-sugar levels and pattern of N-linked glycosylation

Chemical perturbation studies reveal robustness in glycosylation systems, based on comparison of LC-MS/MS quantification of cellular nucleotide-sugar levels with the observed N-linked glycan patterns.