About

Randy Hampton is the principal investigator of the Hampton lab and a Professor in the section of Cell and Developmental Biology within the Division of Biological Sciences at the University of California, San Diego (UCSD). He received his Ph.D. from the University of Wisconsin-Madison and completed his postdoctoral work at the University of California, Berkeley. Randy leads research efforts focused on cellular and developmental biology, with particular attention to mechanisms related to protein degradation and quality control within the endoplasmic reticulum (ER). Outside of his academic pursuits, Randy enjoys playing the banjo.

Research topics

• Biology
• Cell biology
• Biochemistry
• Chemistry

Selected publications

• Exploring the “misfolding problem” by systematic discovery and analysis of functional-but-degraded proteins

  Molecular Biology of the Cell · 2023-09-20 · 2 citations

  articleOpen accessSenior author

  In both health and disease, the ubiquitin-proteasome system (UPS) degrades point mutants that retain partial function but have decreased stability compared with their wild-type counterparts. This class of UPS substrate includes routine translational errors and numerous human disease alleles, such as the most common cause of cystic fibrosis, ΔF508-CFTR. Yet, there is no systematic way to discover novel examples of these "minimally misfolded" substrates. To address that shortcoming, we designed a genetic screen to isolate functional-but-degraded point mutants, and we used the screen to study soluble, monomeric proteins with known structures. These simple parent proteins yielded diverse substrates, allowing us to investigate the structural features, cytotoxicity, and small-molecule regulation of minimal misfolding. Our screen can support numerous lines of inquiry, and it provides broad access to a class of poorly understood but biomedically critical quality-control substrates.

  PublisherOA PDFDOI

• Inner-nuclear-membrane–associated degradation employs Dfm1-independent retrotranslocation and alleviates misfolded transmembrane-protein toxicity

  Molecular Biology of the Cell · 2021-02-10 · 5 citations

  articleOpen accessSenior authorCorresponding

  To undergo degradation, transmembrane ER and inner-nuclear-membrane (INM) proteins are extracted from lipid bilayers in a process known as retrotranslocation. In ERAD-M, retrotranslocation requires Dfm1. We show that INM-associated degradation is Dfm1 independent. Nonetheless, private ER and INM channels constitute a crucial proteostatic network.

  PublisherOA PDFDOI

• Direct involvement of Hsp70 ATP hydrolysis in Ubr1-dependent quality control

  Molecular Biology of the Cell · 2020 · 18 citations

  Senior authorCorresponding
  • Biology
  • Biochemistry
  • Cell biology

  Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone-dependent ubiquitination by the action of the E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70's role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of Sse1, and the action of J-proteins are all needed for Ubr1-mediated quality control. To discern whether chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage in which efficiently folded proteins are spared, while slow-folding or nonfolding proteins are iteratively tagged with ubiquitin for subsequent degradation.

  PublisherOA PDFDOI

• An autonomous, but INSIG-modulated, role for the Sterol Sensing Domain in mallostery-regulated ERAD of yeast HMG-CoA reductase

  bioRxiv (Cold Spring Harbor Laboratory) · 2020-08-21 · 2 citations

  preprintOpen accessSenior authorCorresponding

  Abstract HMG-CoA reductase (HMGR) undergoes feedback regulated degradation as part of sterol pathway control. Degradation of the yeast HMGR isozyme Hmg2 is controlled by the sterol pathway intermediate GGPP, which causes misfolding of Hmg2 to enhance its ERAD by the HRD pathway. GGPP-dependent reversible misfolding of Hmg2 is remarkably similar to classic allosteric control; we recently labeled this process mallostery to fuse the ideas of misfolding and allostery. We have evaluated the role of the Hmg2 sterol sensing domain (SSD) in mallostery, and the involvement of highly conserved INSIG proteins in SSD function. The SSD is a membrane-embedded motif found in many sterol-related proteins. The Hmg2 SSD was critical for in vivo regulated degradation of Hmg2, and required for mallosteric misfolding of GGPP as studied by in vitro limited proteolysis. The Hmg2 SSD functions in mallostery independently of conserved yeast INSIG proteins. However, this autonomous action of the SSD was modulated by INSIG, thus imposing a second layer of control on Hmg2 regulation. SSD-mediated mallostery occurs prior to HRD dependent ubiquitination, defining a pathway regulation involving SSD-mediated misfolding followed by HRD dependent ubiquitination. GGPP dependent misfolding occurred at a much slower rate in the absence of a functional SSD, indicating that the SSD functions to allow physiologically useful rate of GGPP response, and implying that the SSD is not a binding site for GGPP. We used unresponsive Hmg2 SSD mutants to test the importance of quaternary structure in mallosteric regulation: the presence of a non-responsive Hmg2 mutant strongly suppressed regulation of a co-expressed, normal Hmg2. Finally, we have found that GGPP regulated misfolding occurred in detergent solubilized Hmg2, indicating that the mallosteric response is an intrinsic feature of the Hmg2 multimer. The preserved response of Hmg2 when in micellar solution will allow next-level studies on the structural and biophysical features of this novel fusion of regulation and protein quality control.

  PublisherOA PDFDOI

• An autonomous, but INSIG-modulated, role for the sterol sensing domain in mallostery-regulated ERAD of yeast HMG-CoA reductase

  Journal of Biological Chemistry · 2020 · 12 citations

  Senior authorCorresponding
  • Biology
  • Biochemistry
  • Cell biology

  HMG-CoA reductase (HMGR) undergoes feedback-regulated degradation as part of sterol pathway control. Degradation of the yeast HMGR isozyme Hmg2 is controlled by the sterol pathway intermediate GGPP, which causes misfolding of Hmg2, leading to degradation by the HRD pathway; we call this process mallostery. We evaluated the role of the Hmg2 sterol sensing domain (SSD) in mallostery, as well as the involvement of the highly conserved INSIG proteins. We show that the Hmg2 SSD is critical for regulated degradation of Hmg2 and required for mallosteric misfolding of GGPP as studied by in vitro limited proteolysis. The Hmg2 SSD functions independently of conserved yeast INSIG proteins, but its function was modulated by INSIG, thus imposing a second layer of control on Hmg2 regulation. Mutant analyses indicated that SSD-mediated mallostery occurred prior to and independent of HRD-dependent ubiquitination. GGPP-dependent misfolding was still extant but occurred at a much slower rate in the absence of a functional SSD, indicating that the SSD facilitates a physiologically useful rate of GGPP response and implying that the SSD is not a binding site for GGPP. Nonfunctional SSD mutants allowed us to test the importance of Hmg2 quaternary structure in mallostery: a nonresponsive Hmg2 SSD mutant strongly suppressed regulation of a coexpressed, normal Hmg2. Finally, we have found that GGPP-regulated misfolding occurred in detergent-solubilized Hmg2, a feature that will allow next-level analysis of the mechanism of this novel tactic of ligand-regulated misfolding.

  PublisherOA PDFDOI

• HRD Complex Self-Remodeling Enables a Novel Route of Membrane Protein Retrotranslocation

  iScience · 2020 · 22 citations

  Senior authorCorresponding
  • Cell biology
  • Chemistry
  • Biology

  ER-associated degradation (ERAD) targets misfolded ER proteins for degradation. Retrotranslocation, a key feature of ERAD, entails removal of ubiquitinated substrates into the cytosol for proteasomal destruction. Recently, it has been shown that the Hrd1 E3 ligase forms a retrotranslocation channel for luminal (ERAD-L) substrates. Conversely, our studies found that integral membrane (ERAD-M) substrates exit the ER through a distinct pathway mediated by the Dfm1 rhomboid protein. Those studies also revealed a second, Hrd1-dependent pathway of ERAD-M retrotranslocation can arise in dfm1Δ null. Here we show that, in the dfm1Δ null, the HRD complex undergoes remodeling to a form that mediates ERAD-M retrotranslocation. Specifically, Hrd1's normally present stochiometric partner Hrd3 is efficiently removed during suppressive remodeling, allowing Hrd1 to function in this novel capacity. Neither Hrd1 autoubiquitination nor its cytosolic domain is required for suppressive ERAD-M retrotranslocation. Thus, the HRD complex displays remarkable functional flexibility in response to ER stress.

  PublisherOA PDFDOI

• Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration

  Yeast · 2019-05-10 · 21 citations

  articleSenior authorCorresponding

  Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these "ICE" vectors, for "Integrating after CEN Excision." The ICE series was created by introducing a "rare-cutter" NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.

  PublisherDOI

• Assays for protein retrotranslocation in ERAD

  Methods in enzymology on CD-ROM/Methods in enzymology · 2019-01-01 · 10 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• Mallostery: Ligand‐dependent Misfolding as a Strategy for Protein Regulation

  The FASEB Journal · 2019-04-01

  articleSenior author

  Protein quality control is an essential set of processes that allow cells to detect, manage, and destroy misfolded and otherwise aberrant proteins. A substantial component of protein quality control involves the selective destruction of misfolded proteins by the ubiquitin proteasome system. Despite the broad range of substrates subject to quality control destruction, these pathways nevertheless show striking specificity for misfolded versions of otherwise stable proteins. This high selectivity can be exploited to regulate the levels of normal proteins through ligand‐mediated, reversible misfolding. This misfolding appears to be a variant of allosteric control that, instead of causing a change in enzyme function, causes a change in enzyme folding to allow recognition and destruction on a contingency basis. This talk will describe an example of such regulated misfolding involved in the control of the sterol synthesizing mevalonate pathway in yeast. HMG‐CoA Reductase (HMGR) is a rate limiting enzyme of the sterol pathway that undergoes regulated degradation as a mechanism of feedback regulation. In yeast, degradation of the HMGR isozyme Hmg2 is keyed to levels of the 20‐carbon isoprenoid geranylgeranyl pyrophosphate (GGPP). When GGPP levels are high, Hmg2 is efficiently degraded by the HRD pathway, which is a major and highly conserved pathway of quality control in the ER known as ER‐associated degradation (ERAD). We have shown that GGPP causes reversible misfolding of Hmg2 that promotes HRD dependent degradation. GGPP‐regulated Hmg2 misfolding shows remarkable similarities to allosteric regulation. GGPP action on Hmg2 is reversible and highly potent, occurring in the mid‐nanomolar range. GGPP's effects are highly specific for its structure, and its effects on Hmg2 can be antagonized both in vivo and in vitro by a related molecule. These features point to a high potency, ligand‐mediated effect on Hmg2 structure. Consistent with this, Hmg2 exists as a multimer, and unregulated Hmg2 variants show classic “toxic subunit” behavior on the regulation of wild‐type Hmg2. Finally, these effects are antagonized by chemical chaperones, demonstrating that the transition caused by GGPP is consistent with misfolding. Accordingly we call this type of regulation “mallostery” to include the ideas of misfolding and allostery in a single term. When viewed through the lens of mallostery, it appears that a number of highly diverse regulatory situations employ similar molecular tactics. Although the generality remains to be discovered, the broadest possibility is for the design of “mallosteric drugs” that specifically program the destruction of desired targets for clinical or basic scientific benefit. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

  PublisherDOI

• Systematic Gene-to-Phenotype Arrays: A High-Throughput Technique for Molecular Phenotyping

  Molecular Cell · 2018-01-01 · 11 citations

  articleOpen accessCorresponding
  PublisherOA PDFDOI

Recent grants

• Mallosteric Regulation of the Sterol Pathway

  NIH · $5.9M · 1997–2025

• Protein degradation and cholesterol regulation

  NIH · $2.4M · 1997–2021

• Protein degradation and cholesterol regulation

  NIH · $376k · 1997–2021

• NIH Grant R01GM092748

  NIH · $1.1M · 2016

• Protein degradation and cholesterol regulation

  NIH · $328k · 1997–2017

Frequent coauthors

• Sonya E. Neal

  University of California, San Diego

  10 shared

• Renee M. Garza

  Salk Institute for Biological Studies

  10 shared

• Richard G. Gardner

  University of Washington

  8 shared

• Stephen Cronin

  South Tyneside District Hospital

  7 shared

• Margaret A. Wangeline

  University of California, San Diego

  7 shared

• Amanjot Singh

  Credit Valley Hospital

  6 shared

• Nidhi Vashistha

  Malaviya National Institute of Technology Jaipur

  6 shared

• C R Raetz

  Duke Medical Center

  6 shared

Labs

• Hampton LabPI

  The Hampton Lab focuses on the mechanisms of protein quality control and degradation in the endoplasmic reticulum.