Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Huntington's disease is caused by an expanded polyglutamine tract in huntingtin protein, leading to accumulation of huntingtin in the nuclei of striatal neurons. The 18 amino-acid amino-terminus of huntingtin is an amphipathic alpha helical membrane-binding domain that can reversibly target to vesicles and the endoplasmic reticulum (ER). The association of huntingtin to the ER is affected by ER stress. A single point mutation in huntingtin 1-18 predicted to disrupt this helical structure displayed striking phenotypes of complete inhibition of polyglutamine-mediated aggregation, increased huntingtin nuclear accumulation and greatly increased mutant huntingtin toxicity in a striatal-derived mouse cell line. Huntingtin vesicular interaction mediated by 1-18 is specific to late endosomes and autophagic vesicles. We propose that huntingtin has a normal biological function as an ER-associated protein that can translocate to the nucleus and back out in response to ER stress or other events. The increased nuclear entry of mutant huntingtin due to loss of ER-targeting results in increased toxicity.

Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin ␣ (Imp ␣) NLS receptor. Imp ␣ is in turn bound by importin ␤ (Imp ␤), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp ␣ and instead bind directly to Imp ␤. Using in vitro nuclear import assays, we demonstrate that Imp ␣ is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp ␤ proved both sufficient and necessary, in that other ␤-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp ␤ for Imp ␣, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp ␤ is also similar to Imp ␣ binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp ␤ and that therefore function independently of Imp ␣.The majority of nuclear proteins are targeted to the nucleus by basic, generally lysine-rich nuclear localization signals (NLSs) that serve as binding sites for an NLS receptor termed importin ␣ (Imp ␣) or karyopherin ␣ (reviewed in references 29 and 44). Imp ␣ in turn interacts with a second import factor, termed importin ␤ (Imp ␤) or karyopherin ␤1, that mediates docking of the resultant ternary complex to the cytoplasmic face of the nuclear pore complex (NPC) via a direct interaction with specific nucleoporins (5,16,32,38). The subsequent translocation of this heterotrimer through the NPC remains poorly understood but is known to require energy and may be mediated by additional Imp ␤-nucleoporin interactions (39,45). Once the heterotrimer reaches the nuclear face of the NPC, the GTP-bound form of the Ran GTPase directly binds to Imp ␤, resulting in the release of Imp ␣ and the NLS protein into the nucleoplasm (15,25,33). Ran, which is found in the GDP-bound form in the cytoplasm and in the GTP-bound form in the nucleus, is therefore a major determinant of the directionality of nuclear import and may also provide a source of energy (21,31,36,45). Once the NLS protein is released, both Imp ␣ and Imp ␤ are separately recycled back to the cytoplasm, where they can then participate in additional rounds of nuclear import.Human immunodeficiency virus type 1 (HIV-1) encodes two essential regulatory proteins that are both active in the cell nucleus (reviewed in references 8 and 11). The Tat protein is an unusual transcriptional transactivator that dramatically enhances the processivity of transcription directed by the viral long terminal repeat promoter element. Tat funct...