Department of Cell Biology


Sep 2019

Excited to have Ellen Goodall (from Andy Martin's lab at Berkeley) join the lab as a post-doc and Alex Panov join the lab as a graduate student from the BBS program.

May 2019

Excited to have Melissa Hoyer join the lab! Recent alum from Gia Voeltz's lab (UC Boulder).

May 2019

Congratulations to Heeseon and Alban on the publication of their new paper in Molecular Cell identifying a new ER-phagy receptor TEX264. An H, Ordureau A, Paulo JA, Shoemaker CJ, Denic V, Harper JW. TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress. Mol Cell. 2019 Apr 11. pii: S1097-2765(19)30258-8. doi: 10.1016/j.molcel.2019.03.034. [Epub ahead of print]

Nov 2018

Congratulations to Jin-mi, whose paper on TBK1 and RAB7A appeared recently in Science Advances. In this work, we identify S72 in RAB7A as a target of TBK1 in response to mitochondrial depolarization and activation of the PARKIN pathway for mitophagy. Heo et al., RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway. Science Advances (

Apr 2018

Alban's paper describing digital snapshots of PARKIN activity on mitochondria using Parallel Reaction Monitoring is out in Molecular Cell: Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics. Mol Cell. (2018) 70:211-227.

Jan 2018

Our new review on the Parkin-Pink1 pathway is out in a special issue of Nature Reviews - Molecular Cell Biology focused on mitochondrial biology. Check it out!

Dec 2017

Heeseon's paper on ribophagy is now out in Nature Cell Biology...

Autophagy constitutes a major mechanism for recycling of long-lived or damaged proteins. Among the most long-lived and abundant cellular machines in eukaryotes are ribosomes. Multiple quality control mechanisms impinge on ribosome function, including turnover of supernumerary ribosomal subunits and defective nascent chains via the ubiquitin-proteasome system. In contrast, whether and how intact or damaged ribosomes are selectively degraded in order to recover amino acid and nucleic acid building blocks is poorly understood. While nitrogen starvation has been proposed to promote selective autophagic degradation of ribosomes (“ribophagy”) in yeast, it is unclear in mammals: 1) whether ribophagy occurs in a regulated manner, 2) whether ribophagy is promoted through agents that lead to damaged or stalled ribosomes, or particular types of proteotoxic stress, and 3) whether ribosomes and other cytosolic proteins are degraded through parallel pathways in response to proteotoxic stress or agents typically used to induce selective autophagy. Here, we employ genomically encoded mammalian Ribo-Keima reporter cell lines for both large and small ribosomal subunits to systematically quantify ribophagic flux. Ribophagy induced by starvation or mTOR inhibition is VPS34-dependent, but is largely independent of the ATG8 conjugation system. Ribophagy was not induced upon inhibition of translational elongation or nascent chain uncoupling, but was strongly activated upon proteotoxic stress by sodium arsenite and chromosome mis-segregation, dependent upon VPS34 and ATG8 conjugation. When benchmarked against several cytoplasmic Keima reporter proteins, ribophagy was found to be comparatively selective. Interestingly, we found that agents often used to induce selective autophagy also promote ribophagy and autophagic flux of other cytosolic reporters at rates similar to that seen upon induction of bulk autophagy via mTOR inhibition, suggesting widespread “by-stander” degradation during selective autophagy. This study provides the first visualization and quantification of trafficking of ribosomes to the lysosome in mammalian cells, identifies stress agents that promote this process, and demonstrates extensive “by-stander” autophagy during selective autophagy of damaged organelles.

An and Harper (2017) Systematic Analysis of Ribophagy in Human Cells Reveals By-stander Degradation During Selective Autophagy. Nature Cell Biology, online.

Oct 2017

Laura's paper analyzing cells lacking ATG8 proteins and discovering RMC1 as a regulator of CCZ1/MON1 is out in MCB (online)...Check it out:

May 2017

BioPlex 2.0 released.....

The most recent project from the Gygi & Harper Lab’s BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes) project, recently featured in Nature, uses affinity purification-mass spectrometry to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome. It is currently the largest such network assembled, consisting of 56,000 candidate interactions and more than 29,000 previously unknown co-associations.  We have provided a wealth of data related to protein localization, protein domain interactions, complexes enriched in fitness genes, and complexes liked to many human diseases. You can read further about this project here.

Oct 2016

Congratulations to David Rhee in the Harper lab and Danny Scott in the Schulman lab for the recent publication of a paper in Cell describing ARIH1 as an RBR class ubiquitin ligase that works in unison with cullin-RING E3s to control substrate ubiquitylation. This exciting collaboration revealed a new ubiquitin transfer mechanism at play for a subset of CRLs involving CUL1, CUL2, and CUL3 based CRLs.

Oct 2016

Congratulations to Virginia, whose paper on QIL1 mutations in patients with early onset fetal mitochondrial encephalopathy recently appeared in eLife. In this work, which was a collaboration with Manuel Schiff's lab in Paris, we found truncation mutations in QIL1 and demonstrated that cells from patients lack itochondrial cristae junctions, and loss of components of these structures. Cells and tissues from patients also have defects in complex IV activity. 

Jun 2016

Christian's paper on the mitochondrial Unfolded Protein Response (UPR) has now appeared in Nature. Congratulations!

In this paper, we use proteomics and RNA-Seq to describe the cells response to protein misfolding in the mitochondrial matrix. We find large numbers of changes in transcription and protein content in mitochondria after acute mitochondrial misfolding. Surprisingly we found that UPRmt blocks the activity of mitochondrial RNAse P, responsible for processing mitochondrial Pre-RNA and also blocks translation in the matrix. This study provides a framework for understanding mitochondrial UPR.

Dec 2015

Joe and Laura's analysis of NCOA4 and ferritinophagy has appeared in Elife!!! In 2014, we discovered NCOA4 and demonstrated a role for it in the targeting of ferritin to autophagosomes to control iron availability. NCOA4 binds directly to ferritin and targets it to autophagosomes, which facilitates delivery of ferritin to the lysosome wheere its degradation leads to the release of iron. However, how ferritinophagy flux is controlled and the roles of NCOA4 in iron-dependent processes are poorly understood. Through analysis of the NCOA4-FTH1 interaction, we demonstrate that direct association via a key surface arginine in FTH1 and a C-terminal element in NCOA4 is required for delivery of ferritin to the lysosome via autophagosomes. Moreover, NCOA4 abundance is under dual control via autophagy and the ubiquitin proteasome system. Ubiquitin-dependent NCOA4 turnover is promoted by excess iron and involves an iron-dependent interaction between NCOA4 and the HERC2 ubiquitin ligase. In zebrafish and cultured cells, NCOA4 plays an essential role in erythroid differentiation. This work reveals the molecular nature of the NCOA4-ferritin complex and explains how intracellular iron levels modulate NCOA4-mediated ferritinophagy in cells and in an iron-dependent physiological setting.

Ferritinophagy via NCOA4 is required for erythropoiesis and is regulated by iron dependent HERC2-mediated proteolysis. Mancias JD, Pontano Vaites L, Nissim S, Biancur DE, Kim AJ, Wang X, Liu Y, Goessling W, Kimmelman AC, Harper JW.

Elife. 2015 Oct 5;4. doi: 10.7554/eLife.10308.
Oct 2015

Jin-mi and Alban's new analysis of the role of autophagy adaptors in mitophagy has appeared in Molecular Cell!!

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson’s disease and amyotrophic lateral sclerosis in a common selective autophagy pathway. 

The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy.

Jin-Mi Heo, Alban Ordureau, Joao A. Paulo, Jesse Rinehart, J. Wade Harper 

Molecular Cell60:7-20 (2015). 

also see: 

Oct 2015

Mali's work defining the p97 adaptor protein network and discovery of a role for p97 in ciliagenesis has appeared!!

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to ‘segregate’ ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left–right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP–UBXN10 in ciliogenesis.

Systematic proteomics of the VCP–UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis

Malavika Raman, Mikhail Sergeev, Maija Garnaas, John R. Lydeard, Edward L. Huttlin, Wolfram Goessling, Jagesh V. Shah and J.Wade Harper

Nature Cell Biology 17:1356-1369.

Jul 2015

For the last 3 years, the Harper and Gygi labs have been working to build a platform for interaction proteomics that can be used to provide a first pass analysis of the majority of protein coding genes present in the ORFEOME collection, a project co-funded by NHGRI and Biogen. This platform, which involves stable lentiviral expression of proteins in HEK293T cells, allows ~600 baits to be analyzed per month. We also developed a modified version of the CompPASS method for analyzing large scale interaction data, which we call CompPASS-Plus. The first fruits of this have now been published [Huttlin EL, et al. The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell. 2015 162:425-440]. This is an analysis of the first 2500 bait proteins chosen randomly from the ORFEOME. This paper provides a detailed analysis of this interactome, its structure, and what we have learned, including domain linkage and the use of such interactome data sets to inform biological analysis. As part of the NHGRI funded mechanism, we are depositing unpublished data into the BIOGRID went site routinely, and have now deposited more than 5000 bait proteins and more than 50K interactions into this database. We have also created a website for BIOPLEX that allows access to all of the data, including all proteomic data []. This work is continuing and will be routinely updated, with the goal of providing a first pass analysis of the entire ORFEOME over the coming years.

May 2015

Our lab has aninterest in the identification of novel complexes in mitochondria. This is being pursued by Virginia Guarani, a post-doctoral fellow in the lab, and part of her work has recently been published in eLIFE. The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly and degradation of MICOS associated proteins MIC10, MIC26, and MIC27. Additionally, we demonstrated thatin QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and with SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology and mitochondrial function and provides a resource for further analysis of MICOS architecture.

QIL1 is a Novel Mitochondrial Protein Required for MICOS Complex Stability and Cristae Morphology

Virginia Guarani, Elizabeth M. McNeill, Joao A. Paulo, Edward L. Huttlin, Florian Fröhlich, Steven P. Gygi, David Van Vactor, and J. Wade Harper. 

eLIFE, published online.

May 2015

The PTEN-inducible putative kinase protein (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and non-canonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved serine in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using “UBS65A-replacement”, Alban Ordureau and Jin-mi Heo find that PARKIN phosphorylation and activation, and ubiquitylation of lysine residues on a cohort of MOM proteins, occurs similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, poly-UB chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UBS65A-replacement cells. Analogous experiments examining roles of individual UB chain linkage-types revealed importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optinuerin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains and <0.16% the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. We also collaborated with Benda Schulman and Dave Duda to examine the biochemical mechanism of PARKIN activation by phospho-ubiquitin. They showed that combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of non-phosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase, and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains. 

These studies set the stage for a more detailed analysis of the PARKIN system using quantitative proteomics.

Ordureau, A., Heo, J.-M., Duda, D.M., Olszewski J.L., et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc Natl Acad Sci USA, early edition on line.

May 2015

Congratulations to Joe Mancias who was awarded 2015 Burroughs Wellcome Fund Career Award for Medical Scientist!!!!

Also, congratulations to Sharan Swarup who received a post-doctoral fellowship from Candadian Health Services!!!!

This is great news....

Oct 2014

PINK1 and PARKIN – two proteins mutated in early onset Parkinson’s Disease - are known to function in a signaling cascade that leads to ubiquitylation of mitochondrial outer membrane proteins on damaged mitochondria, but the precise mechanism through by which PINK1 activates PARKIN ubiquitin ligase activity and retention on the mitochondrial membrane is poorly understood. In this work, Alban Ordureau in the lab used quantitative proteomics and a technique called ubiquitin AQUA to examine the kinetics and specificity of PINK1 and PARKIN-dependent ubiquitin chain synthesis on damaged mitochondria in vivo. Through mechanistic and biochemical analysis, as well as live-cell imaging performed by Shireen Sarraf when she was a student in the lab and biophysical experiments done in collaboration with Brenda Schulman, we define multiple steps in the process, revealing a feed-forward mechanism for PARKIN activation and retention on mitochondria. PINK1 phosphorylation of S65 in the UBL of PARKIN leads to activation of its ubiquitin ligase activity by 2400-fold, which in turn promotes the initial synthesis of K6, K11, K48, and K63 ubiquitin chains on mitochondria by PARKIN. Ubiquitin units within newly synthesized ubiquitin chains are then phosphorylated by PINK1 on S65, the residue homologous to S65 in the UBL of PARKIN. This, in turn, serves as a binding site (Kd = 17 nM) for activated PARKIN, leading to retention of PARKIN on poly-ubiquitinated mitochondria, which could support both further ubiquitin chain synthesis and recruitment of proteins to promote mitophagy.  Our data reveal a feed-forward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage. This work also provides a framework for quantitative analysis of phosphorylation and ubiquitin dependent signaling systems in vitro and in vivo. Congratulations Alban!

Ordureau A, Sarraf SA, Duda DM, Heo J-M, Jedrychowski MP, Sviderskiy V, Olszewski JL, Koerber JT, Xie T, Beausoleil SA, Wells JA, Gygi SP, Schulman BA, and Harper JW (2014) Quantitative proteomics reveal a feed-forward mechanism for mitochondrial PARKIN translocation and UB chain synthesis. Molecular Cell, 56: 360–375.